Vertebrate embryonic pattern-inducing proteins, and uses related thereto

ABSTRACT

The present invention concerns the discovery that proteins encoded by a family of vertebrate genes, termed here hedgehog-related genes, comprise morphogenic signals produced by embryonic patterning centers, and are involved in the formation of ordered spatial arrangements of differentiated tissues in vertebrates. The present invention makes available compositions and methods that can be utilized, for example to generate and/or maintain an array of different vertebrate tissue both in vitro and in vivo.

RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. Ser. No.08/435,093, filed May 4, 1995, which is a continuation-in-part of U.S.Ser. No. Ser. No. 08/356,060, filed Dec. 14, 1994, which is acontinuation-in-part of U.S. Ser. No. Ser. No. 08/227,371 filed Dec. 30,1993 and entitled “Vertebrate Embryonic Pattern-Inducing Proteins andUses Related Thereto”, the teachings of which are incorporated herein byreference.

FUNDING

[0002] Work described herein was supported by funding from the NationalInstitutes of Health. The United States Government has certain rights inthe invention.

BACKGROUND OF THE INVENTION

[0003] Pattern formation is the activity by which embryonic cells formordered spatial arrangements of differentiated tissues. The physicalcomplexity of higher organisms arises during embryogenesis through theinterplay of cell-intrinsic lineage and cell-extrinsic signaling.Inductive interactions are essential to embryonic patterning invertebrate development from the earliest establishment of the body plan,to the patterning of the organ systems, to the generation of diversecell types during tissue differentiation (Davidson, E., (1990)Development 108: 365-389; Gurdon, J. B., (1992) Cell 68: 185-199;Jessell, T. M. et al., (1992) Cell 68: 257-270). The effects ofdevelopmental cell interactions are varied. Typically, responding cellsare diverted from one route of cell differentiation to another byinducing cells that differ from both the uninduced and induced states ofthe responding cells (inductions). Sometimes cells induce theirneighbors to differentiate like themselves (homoiogenetic induction); inother cases a cell inhibits its neighbors from differentiating likeitself. Cell interactions in early development may be sequential, suchthat an initial induction between two cell types leads to a progressiveamplification of diversity. Moreover, inductive interactions occur notonly in embryos, but in adult cells as well, and can act to establishand maintain morphogenetic patterns as well as induce differentiation(J. B. Gurdon (1992) Cell 68:185-199).

[0004] The origin of the nervous system in all vertebrates can be tracedto the end of gastrulation. At this time, the ectoderm in the dorsalside of the embryo changes its fate from epidermal to neural. The newlyformed neuroectoderm thickens to form a flattened structure called theneural plate which is characterized, in some vertebrates, by a centralgroove (neural groove) and thickened lateral edges (neural folds). Atits early stages of differentiation, the neural plate already exhibitssigns of regional differentiation along its anterior posterior (A-P) andmediolateral axis (M-L). The neural folds eventually fuse at the dorsalmidline to form the neural tube which will differentiate into brain atits anterior end and spinal cord at its posterior end. Closure of theneural tube creates dorsal/ventral differences by virtue of previousmediolateral differentiation. Thus, at the end of neurulation, theneural tube has a clear anterior-posterior (A-P), dorsal ventral (D-V)and mediolateral (M-L) polarities (see, for example, Principles inNeural Science (3rd), eds. Kandel, Schwartz and Jessell, ElsevierScience Publishing Company: NY, 1991; and Developmental Biology (3rd),ed. S. F. Gilbert, Sinauer Associates: Sunderland Mass., 1991).Inductive interactions that define the fate of cells within the neuraltube establish the initial pattern of the embryonic vertebrate nervoussystem. In the spinal cord, the identify of cell types is controlled, inpart, by signals from two midline cell groups, the notochord and floorplate, that induce neural plate cells to differentiate into floor plate,motor neurons, and other ventral neuronal types (van Straaten et al.(1988) Anat. Embryol. 177:317-324; Placzek et al. (1993) Development117:205-218; Yamada et al. (1991) Cell 64:035-647; and Hatta et al.(1991) Nature 350:339-341). In addition, signals from the floor plateare responsible for the orientation and direction of commissural neuronoutgrowth (Placzek, M. et al., (1990) Development 110: 19-30). Besidespatterning the neural tube, the notochord and floorplate are alsoresponsible for producing signals which control the patterning of thesomites by inhibiting differentiation of dorsal somite derivatives inthe ventral regions (Brand-Saberi, B. et al., (1993) Anat. Embryol. 188:239-245; Porquie, O. et al., (1993) Proc. Natl. Acad. Sci. USA 90:5242-5246).

[0005] Another important signaling center exists in the posteriormesenchyme of developing limb buds, called the Zone of PolarizingActivity, or “ZPA”. When tissue from the posterior region of the limbbud is grafted to the anterior border of a second limb bud, theresultant limb will develop with additional digits in a mirror-imagesequence along the anteroposterior axis (Saunders and Gasseling, (1968)Epithelial-Mesenchymal Interaction, pp. 78-97). This finding has led tothe model that the ZPA is responsible for normal anteroposteriorpatterning in the limb. The ZPA has been hypothesized to function byreleasing a signal, termed a “morphogen”, which forms a gradient acrossthe early embryonic bud. According to this model, the fate of cells atdifferent distances from the ZPA is determined by the localconcentration of the morphogen, with specific thresholds of themorphogen inducing successive structures (Wolpert, (1969) Theor. Biol.25:1-47). This is supported by the finding that the extent of digitduplication is proportional to the number of implanted ZPA cells(Tickle, (1981) Nature 254:199-202).

[0006] A candidate for the putative ZPA morphogen was identified by thediscovery that a source of retinoic acid can result in the same type ofmirror-image digit duplications when placed in the anterior of a limbbud (Tickle et al., (1982) Nature 296:564-565; Summerbell, (1983) J.Embryol 78:269-289). The response to exogenous retinoic acid isconcentration dependent as the morphogen model demands (Tickle et al.,(1985) Dev. Biol. 109:82-95). Moreover, a differential distribution ofretinoic acid exists across the limb bud, with a higher concentration inthe ZPA region (Thaller and Eichele, (1987) Nature 327:625-628).

[0007] Recent evidence, however, has indicated that retinoic acid isunlikely to be the endogenous factor responsible for ZPA activity(reviewed in Brockes, (1991) Nature 350:15; Tabin, (1991) Cell66:199-217). It is now believed that rather than directly mimicking anendogenous signal, retinoic acid implants act by inducing an ectopicZPA. The anterior limb tissue just distal to a retinoic acid implant anddirectly under the ectoderm has been demonstrated to acquire ZPAactivity by serially transplanting that tissue to another limb bud(Summerbell and Harvey, (1983) Limb Development and Regeneration pp.109-118; Wanek et al., (1991) Nature 350:81-83). Conversely, the tissuenext to a ZPA graft does not gain ZPA activity (Smith, (1979) J. Embryol52:105-113). Exogenous retinoic acid would thus appear to act upstreamof the ZPA in limb patterning.

[0008] The immediate downstream targets of ZPA action are not known.However, one important set of genes which are ectopically activatedduring ZPA-induced pattern duplications are the 5′ genes of the Hoxdcluster. These genes are normally expressed in a nested patternemanating from the posterior margin of the limb bud (Dolle et al.,(1989) Nature 342:767-772; Izpisua-Belmonte et al., (1991) Nature350:585-589). This nested pattern of Hox gene expression has beendirectly demonstrated to determine the identity of the structuresproduced along the anteroposterior axis of the limb (Morgan et al.,(1993) Nature 358:236-239). As this would predict, ZPA grafts whichproduce mirror-image duplication of structures at an anatomical levelfirst lead to the ectopic activation of the Hoxd genes in a mirror-imageduplication at the molecular level. (Nohno et al., (1991) Cell64:1197-1205; Izpisua-Belmonte et al., (1991) Nature 350:585-589). Themolecular signals which regulate the expression of these important genesare currently not understood.

SUMMARY OF THE INVENTION

[0009] The present invention relates to the discovery of a novel familyof genes, and gene products, expressed in vertebrate organisms, whichgenes referred to hereinafter as the “hedgehog” gene family, theproducts of which are referred to as hedgehog proteins. The products ofthe hedgehog gene have apparent broad involvement in the formation andmaintenance of ordered spatial arrangements of differentiated tissues invertebrates, both adult and embryonic, and can be used to generateand/or maintain an array of different vertebrate tissue both in vitroand in vivo.

[0010] In general, the invention features hedgehog polypeptides,preferably substantially pure preparations of one or more of the subjecthedgehog polypeptides. The invention also provides recombinantlyproduced hedgehog polypeptides. In preferred embodiments the polypeptidehas a biological activity including: an ability to modulateproliferation, survival and/or differentiation of mesodermally-derivedtissue, such as tissue derived from dorsal mesoderm; the ability tomodulate proliferation, survival and/or differentiation ofectodermally-derived tissue, such as tissue derived from the neuraltube, neural crest, or head mesenchyme; the ability to modulateproliferation, survival and/or differentiation of endodermally-derivedtissue, such as tissue derived from the primitive gut. Moreover, inpreferred embodiments, the subject hedgehog proteins have the ability toinduce expression of secondary signaling molecules, such as members ofthe Transforming Growth Factor β family, as well as members of thefibroblast growth factor (FGF) family.

[0011] In a preferred embodiment, the polypeptide is identical with orhomologous to a Sonic hedgehog (Shh) polypeptide, such as a mammalianShh represented by SEQ ID Nos:13 or 11, an avian Shh represented by SEQID No: 8, or a fish Shh represented by SEQ ID No: 12. For instance, theShh polypeptide preferably has an amino acid sequence at least 60%homologous to a polypeptide represented by any of SEQ ID Nos: 8, 11, 12or 13, though polypeptides with higher sequence homologies of, forexample, 80%, 90% or 95% are also contemplated. Exemplary Shh proteinsare represented by SEQ ID No. 40. The Shh polypeptide can comprise afull length protein, such as represented in the sequence listings, or itcan comprise a fragment of, for instance, at least 5, 10, 20, 50, 100,150 or 200 amino acids in length. Preferred hedgehog polypeptidesinclude Shh sequences corresponding approximately to the naturalproteolytic fragments of the hedgehog proteins, such as from aboutCys-24 through about the region that contains the proteolytic processingsite, e.g., Ala-194 to Gly-203, or from about Cys-198 through Ala-475 ofthe human Shh protein, or analogous fragments thereto.

[0012] In another preferred embodiment, the polypeptide is identicalwith or homologous to an Indian hedgehog (Ihh) polypeptide, such as ahuman Ihh represented by SEQ ID No:14, or a mouse Ihh represented by SEQID No: 10. For instance, the Ihh polypeptide preferably has an aminoacid sequence at least 60% homologous to a polypeptide represented byeither of SEQ ID Nos: 10 or 14, though Ihh polypeptides with highersequence homologies of, for example, 80%, 90% or 95% are alsocontemplated. The polypeptide can comprise the full length proteinrepresented by in part by these sequences, or it can comprise a fragmentof, for instance, at least 5, 10, 20, 50, 100, 150 or 200 amino acids inlength. Preferred Ihh polypeptides comprise an N-terminal fragment fromCys-28 through the region that contains the proteolytic processing site,e.g., Ala-198 to Gly-207, or a C-terminal fragment from about Cys-203through Ser-411 of the mouse Ihh represented by SEQ ID No:10, oranalogous fragments thereto.

[0013] In still a further preferred embodiment, the polypeptide isidentical with or homologous to a Desert hedgehog (Dhh) polypeptide,such as a mouse Dhh represented by SEQ ID No: 9. For instance, the Dhhpolypeptide preferably has an amino acid sequence at least 60%homologous to a polypeptide represented by SEQ ID No: 9, though Dhhpolypeptides with higher sequence homologies of, for example, 80%, 90%or 95% are also contemplated. The polypeptide can comprise the fulllength protein represented by this sequence, or it can comprise afragment of, for instance, at least 5, 10, 20, 50, 100, 150 or 200 aminoacids in length. Preferred Dhh polypeptides comprise Dhh sequencescorresponding to the N-terminal portion of the protein from about Cys-23through about the region that contains the proteolytic processing site,e.g., Val-124 to Asn-203 or C-terminal fragment from about Cys-199through Gly-396 of SEQ ID No:9, or analogous fragments thereto.

[0014] In another preferred embodiment, the invention features apurified or recombinant polypeptide fragment of a hedgehog protein,which polypeptide has the ability to modulate, e.g., mimic orantagonize, a the activity of a wild-type hedgehog protein. Preferably,the polypeptide fragment comprises a sequence identical or homologous toan amino acid sequence designated in one of SEQ ID No:8, SEQ ID No:9,SEQ ID No:10, SEQ ID No:11, SEQ ID No:12, SEQ ID No:13, or SEQ ID No:14.More preferably, the polypeptide fragment comprises an amino acidsequence designated in SEQ ID No: 40, e.g., includes the fragment ofCys-1 to Gly-174.

[0015] In yet another preferred embodiment, the invention features apurified or recombinant polypeptide, which polypeptide has a molecularweight of approximately 19 kDa and has the ability to modulate, e.g.,mimic or antagonize, a the activity of a wild-type hedgehog protein.Preferably, the polypeptide comprises an amino acid sequence identicalor homologous to an sequence designated in one of SEQ ID No:8, SEQ IDNo:9, SEQ ID No:10, SEQ ID No:11, SEQ ID No:12, SEQ ID No:13, or SEQ IDNo:14. More preferably, the polypeptide comprises an amino acid sequencedesignated in SEQ ID No:40.

[0016] In still another preferred embodiment, the invention features apurified or recombinant hedgehog polypeptide comprising an amino acidsequence represented by the formula A-B wherein, A represents all or theportion of the amino acid sequence designated by residues 1-168 of SEQID No:40; and B represents at least one amino acid residue of the aminoacid sequence designated by residues 169-221 of SEQ ID No:40; wherein Aand B together represent a contiguous polypeptide sequence representedby SEQ ID No:40, and the polypeptide modulates, e.g., mimics orantagonizes, the biological activity of a hedgehog protein. Preferably,B can represent at least 5, 10 or 20 amino acid residues of the aminoacid sequence designated by residues 169-221 of SEQ ID No:40.

[0017] In another embodiment, the invention features a purified orrecombinant polypeptide comprising an amino acid sequence represented bythe formula A-B, wherein A represents all or the portion of the aminoacid sequence designated by residues 24-193 of SEQ ID No:13; and Brepresents at least one amino acid residue of the amino acid sequencedesignated by residues 194-250 of SEQ ID No:13; wherein A and B togetherrepresent a contiguous polypeptide sequence designated in SEQ ID No:13,and the polypeptide modulates, e.g., mimics or antagonizes, thebiological activity of a hedgehog protein.

[0018] In yet another preferred embodiment, the invention features apurified or recombinant polypeptide comprising an amino acid sequencerepresented by the formula A-B, wherein A represents all or the portionof the amino acid sequence designated by residues 25-193, or analogousresidues thereof, of a vertebrate hedgehog polypeptide identical orhomologous to SEQ ID No:11; and B represents at least one amino acidresidue of the amino acid sequence designated by residues 194-250, oranalogous residues thereof, of a vertebrate hedgehog polypeptideidentical or homologous to SEQ ID No:11; wherein A and B togetherrepresent a contiguous polypeptide sequence designated in SEQ ID No:11,and the polypeptide modulates, e.g., agonizes or antagonizes, thebiological activity of a hedgehog protein.

[0019] In another embodiment, the invention features a purified orrecombinant polypeptide comprising an amino acid sequence represented bythe formula A-B, wherein A represents all or the portion of the aminoacid sequence designated by residues 23-193 of SEQ ID No:9; and Brepresents at least one amino acid residue of the amino acid sequencedesignated by residues 194-250 of SEQ ID No:9; wherein A and B togetherrepresent a contiguous polypeptide sequence designated in SEQ ID No:9,and the polypeptide modulates, e.g., agonizes or antagonizes, thebiological activity of a hedgehog protein.

[0020] In yet another embodiment, the invention features a purified orrecombinant polypeptide comprising an amino acid sequence represented bythe formula A-B, wherein A represents all or the portion of the aminoacid sequence designated by residues 28-197 of SEQ ID No:10; and Brepresents at least one amino acid residue of the amino acid sequencedesignated by residues 198-250 of SEQ ID No:10; wherein A and B togetherrepresent a contiguous polypeptide sequence designated in SEQ ID No:10,and the polypeptide modulates, e.g., agonizes or antagonizes, thebiological activity of a hedgehog protein.

[0021] In yet a further preferred embodiment, the invention features apurified or recombinant polypeptide comprising an amino acid sequencerepresented by the formula A-B, wherein A represents all or the portionof the amino acid sequence designated by residues 1-98, or analogousresidues thereof, of a vertebrate hedgehog polypeptide identical orhomologous to SEQ ID No:14; and B represents at least one amino acidresidue of the amino acid sequence designated by residues 99-150, oranalogous residues thereof, of a vertebrate hedgehog polypeptideidentical or homologous to SEQ ID No:14; wherein A and B togetherrepresent a contiguous polypeptide sequence designated in SEQ ID No:14,and the polypeptide modulates, e.g., agonizes or antagonizes, thebiological activity of a hedgehog protein.

[0022] In another preferred embodiment, the invention features a nucleicacid encoding a polypeptide fragment of a hedgehog protein, e.g. afragment described above. Preferably, the polypeptide fragment comprisesan amino acid sequence identical or homologous with a sequencedesignated in one of SEQ ID No:8, SEQ ID No:9, SEQ ID No:10, SEQ IDNo:11, SEQ ID No:12, SEQ ID No:13, or SEQ ID No:14. More preferably, thepolypeptide fragment comprises an amino acid sequence designated in SEQID No:40.

[0023] In yet another preferred embodiment, the invention features anucleic acid encoding a polypeptide, which polypeptide has a molecularweight 19 kDa and has the ability to modulate, e.g., either mimic orantagonize, at least a portion of the activity of a wild-type hedgehogprotein. Preferably, the polypeptide comprises an amino acid sequenceidentical or homologous with a sequence designated in one of SEQ IDNo:8, SEQ ID No:9, SEQ ID No:10, SEQ ID No:11, SEQ ID No:12, SEQ IDNo:13, or SEQ ID No:14. More preferably, the polypeptide comprises anamino acid sequence designated in the general formula SEQ ID No:40.

[0024] In another preferred embodiment, the invention feature a nucleicacid which encodes a polypeptide that modulates, e.g., mimics orantagonizes, the biological activity of a hedgehog protein, whichnucleic acid comprises all or a portion of the nucleotide sequence ofthe coding region of a gene identical or homologous to the nucleotidesequence designated by one of SEQ ID No:1, SEQ ID No:2, SEQ ID No:3, SEQID No:4, SEQ ID No:5, SEQ ID No:6 or SEQ ID No:7. Preferably, thenucleic acid comprises a hedgehog-encoding portion that hybridizes understringent conditions to a coding portion of one or more of the nucleicacids designated by SEQ ID No: 1-7.

[0025] Moreover, as described below, the hedgehog polypeptide can beeither an agonist (e.g. mimics), or alternatively, an antagonist of abiological activity of a naturally occurring form of the protein, e.g.,the polypeptide is able to modulate differentiation and/or growth and/orsurvival of a cell responsive to authentic hedgehog proteins. Homologsof the subject hedgehog proteins include versions of the protein whichare resistant to proteolytic cleavage, as for example, due to mutationswhich alter potential cleavage sequences or which inactivate from a cellwith no further proteolytic cleavage required beyond cleavage of asignal sequence, e.g., truncated forms of the protein, such ascorresponding to the natural proteolytic fragments described below.

[0026] The hedgehog polypeptides of the present invention can beglycosylated, or conversely, by choice of the expression system or bymodification of the protein sequence to preclude glycosylation, reducedcarbohydrate analogs can also be provided. Glycosylated forms includederivatization with glycosaminoglycan chains. Likewise, hedgehogpolypeptides can be generated which lack an endogenous signal sequence(though this is typically cleaved off even if present in the pro-form ofthe protein).

[0027] The subject proteins can also be provided as chimeric molecules,such as in the form of fusion proteins. For instance, the hedgehogprotein can be provided as a recombinant fusion protein which includes asecond polypeptide portion, e.g., a second polypeptide having an aminoacid sequence unrelated (heterologous) to the hedgehog polypeptide, e.g.the second polypeptide portion is glutathione-S-transferase, e.g. thesecond polypeptide portion is an enzymatic activity such as alkalinephosphatase, e.g. the second polypeptide portion is an epitope tag.

[0028] Yet another aspect of the present invention concerns an immunogencomprising a hedgehog polypeptide in an immunogenic preparation, theimmunogen being capable of eliciting an immune response specific for ahedgehog polypeptide; e.g. a humoral response, e.g. an antibodyresponse; e.g. a cellular response. In preferred embodiments, theimmunogen comprising an antigenic determinant, e.g. a uniquedeterminant, from a protein represented by one of SEQ ID Nos. 8-14.

[0029] A still further aspect of the present invention featuresantibodies and antibody preparations specifically reactive with anepitope of the hedgehog immunogen.

[0030] In another preferred embodiment, the invention features a nucleicacid encoding a polypeptide fragment of a hedgehog protein, e.g. afragment described above. Preferably, the polypeptide fragment comprisesan amino acid sequence identical or homologous with a sequencedesignated in one of SEQ ID No:8, SEQ ID No:9, SEQ ID No:10, SEQ IDNo:11, SEQ ID No:12, SEQ ID No:13, or SEQ ID No:14. More preferably, thepolypeptide fragment comprises an amino acid sequence designated in SEQID No:40.

[0031] In yet another preferred embodiment, the invention features anucleic acid encoding a polypeptide, which polypeptide has a molecularweight of approximately 19 kDa and has the ability to modulate, e.g.,either mimic or antagonize, atleast a portion of the activity of awild-type hedgehog protein. Preferably, the polypeptide comprises anamino acid sequence identical or homologous with a sequence designatedin one of SEQ ID No:8, SEQ ID No:9, SEQ ID No:10, SEQ ID No:11, SEQ IDNo:12, SEQ ID No:13, or SEQ ID No:14. More preferably, the polypeptidecomprises an amino acid sequence designated in the general formula SEQID No:40.

[0032] In another preferred embodiment, the invention feature a nucleicacid which encodes a polypeptide that modulates, e.g., mimics orantagonizes, the biological activity of a hedgehog protein, whichnucleic acid comprises all or a portion of the nucleotide sequence ofthe coding region of a gene identical or homologous to the nucleotidesequence designated by one of SEQ ID No:1, SEQ ID No:2, SEQ ID No:3, SEQID No:4, SEQ ID No:5, SEQ ID No:6 or SEQ ID No:7. Preferably, thenucleic acid comprises a hedgehog-encoding portion that hybridizes understringent conditions to a coding portion of one or more of the nucleicacids designated by SEQ ID No:1-7.

[0033] Another aspect of the present invention provides a substantiallyisolated nucleic acid having a nucleotide sequence which encodes ahedgehog polypeptide. In preferred embodiments, the encoded polypeptidespecifically mimics or antagonizes inductive events mediated bywild-type hedgehog proteins. The coding sequence of the nucleic acid cancomprise a sequence which is identical to a coding sequence representedin one of SEQ ID Nos: 1-7, or it can merely be homologous to one or moreof those sequences. For instance, the hedgehog encoding sequencepreferably has a sequence at least 60% homologous to a nucleotidesequence in one or more of SEQ ID Nos: 1-7, though higher sequencehomologies of, for example, 80%, 90% or 95% are also contemplated. Thepolypeptide encoded by the nucleic acid can comprise an amino acidsequence represented in one of SEQ ID Nos: 8-14 such as one of thosefull length proteins, or it can comprise a fragment of that nucleicacid, which fragment may, for instance, encode a fragment which is, forexample, at least 5, 10, 20, 50 or 100 or 200 amino acids in length. Thepolypeptide encoded by the nucleic acid can be either an agonist (e.g.mimics), or alternatively, an antagonist of a biological activity of anaturally occurring form of a hedgehog protein.

[0034] Furthermore, in certain preferred embodiments, the subjecthedgehog nucleic acid will include a transcriptional regulatorysequence, e.g. at least one of a transcriptional promoter ortranscriptional enhancer sequence, which regulatory sequence is operablylinked to the hedgehog gene sequence. Such regulatory sequences can beused in to render the hedgehog gene sequence suitable for use as anexpression vector.

[0035] In yet a farther preferred embodiment, the nucleic acidhybridizes under stringent conditions to a nucleic acid probecorresponding to at least 12 consecutive nucleotides of either sense orantisense sequence of one or more of SEQ ID Nos:1-7; though preferablyto at least 20 consecutive nucleotides; and more preferably to at least40, 50 or 75 consecutive nucleotides of either sense or antisensesequence of one or more of SEQ ID Nos:1-7.

[0036] The invention also features transgenic non-human animals, e.g.mice, rats, rabbits, chickens, frogs or pigs, having a transgene, e.g.,animals which include (and preferably express) a heterologous form of ahedgehog gene described herein, or which misexpress an endogenoushedgehog gene, e.g., an animal in which expression of one or more of thesubject hedgehog proteins is disrupted. Such a transgenic animal canserve as an animal model for studying cellular and tissue disorderscomprising mutated or mis-expressed hedgehog alleles or for use in drugscreening.

[0037] The invention also provides a probe/primer comprising asubstantially purified oligonucleotide, wherein the oligonucleotidecomprises a region of nucleotide sequence which hybridizes understringent conditions to at least 10 consecutive nucleotides of sense orantisense sequence of SEQ ID No:1, or naturally occurring mutantsthereof. Nucleic acid probes which are specific for each of the classesof vertebrate hedgehog proteins are contemplated by the presentinvention, e.g. probes which can discern between nucleic acid encodingan Shh versus an Ihh versus a Dhh versus an Mhh. In preferredembodiments, the probe/primer further includes a label group attachedthereto and able to be detected. The label group can be selected, e.g.,from a group consisting of radioisotopes, fluorescent compounds,enzymes, and enzyme co-factors. Probes of the invention can be used as apart of a diagnostic test kit for identifying dysfunctions associatedwith mis-expression of a hedgehog protein, such as for detecting in asample of cells isolated from a patient, a level of a nucleic acidencoding a subject hedgehog protein; e.g. measuring a hedgehog mRNAlevel in a cell, or determining whether a genomic hedgehog gene has beenmutated or deleted. These so called “probes/primers” of the inventioncan also be used as a part of “antisense” therapy which refers toadministration or in situ generation of oligonucleotide probes or theirderivatives which specifically hybridize (e.g. bind) under cellularconditions, with the cellular mRNA and/or genomic DNA encoding one ormore of the subject hedgehog proteins so as to inhibit expression ofthat protein, e.g. by inhibiting transcription and/or translation.Preferably, the oligonucleotide is at least 10 nucleotides in length,though primers of 20, 30, 50, 100, or 150 nucleotides in length are alsocontemplated.

[0038] In yet another aspect, the invention provides an assay forscreening test compounds for inhibitors, or alternatively, potentiators,of an interaction between a hedgehog protein and a hedgehog receptor. Anexemplary method includes the steps of (i) combining a hedgehogreceptor, either soluble or membrane bound (including whole cells), ahedgehog polypeptide, and a test compound, e.g., under conditionswherein, but for the test compound, the hedgehog protein and thehedgehog receptor are able to interact; and (ii) detecting the formationof a complex which includes the hedgehog protein and the receptor eitherby directly quantitating the complex or by measuring inductive effectsof the hedgehog protein. A statistically significant change, such as adecrease, in the formation of the complex in the presence of a testcompound (relative to what is seen in the absence of the test compound)is indicative of a modulation, e.g., inhibition, of the interactionbetween the hedgehog protein and the receptor.

[0039] Yet another aspect of the present invention concerns a method formodulating one or more of growth, differentiation, or survival of amammalian cell responsive to hedgehog induction. In general, whethercarries out in vivo, in vitro, or in situ, the method comprises treatingthe cell with an effective amount of a hedgehog polypeptide so as toalter, relative to the cell in the absence of hedgehog treatment, atleast one of (i) rate of growth, (ii) differentiation, or (iii) survivalof the cell. Accordingly, the method can be carried out withpolypeptides mimics the effects of a naturally-occurring hedgehogprotein on the cell, as well as with polypeptides which antagonize theeffects of a naturally-occurring hedgehog protein on said cell. Inpreferred embodiments, the hedgehog polypeptide provided in the subjectmethod are derived from verterbrate sources, e.g., are vertebratehedgehog polypeptides. For instance, preferred polypeptides includes anamino acid sequence identical or homologous to an amino acid sequence(e.g., including bioactive fragments) designated in one of SEQ ID No:8,SEQ ID No:9, SEQ ID No:10, SEQ ID No:11, SEQ ID No:12, SEQ ID No:13 orSEQ ID No:14. Furthermore, the present invention contemplates the use ofinvertebrate hedgehog polypeptides, such as the Dros-HH polypeptidedesignated by SEQ ID No:34, or bioactive fragments thereof equivalent tothe subject vertebrate fragments.

[0040] In one embodiment, the subject method includes the treatment oftesticular cells, so as modulate spermatogenesis. In another embodiment,the subject method is used to modulate osteogenesis, comprising thetreatment of osteogenic cells with a hedgehog polypeptide. Liekwise,where the treated cell is a chondrogenic cell, the present method isused to modulate chondrogenesis. In still another embodiment, hedgehogpolypeptides can be used to modulate the differentiation of neuralcells, e.g., the method can be used to cause differentiation of aneuronal cell, to maintain a neuronal cell in a differentiated state,and/or to enhance the survival of a neuronal cell, e.g., to preventapoptosis or other forms of cell death. For instance, the present methodcan be used to affect the differentiation of such neuronal cells asmotor neurons, cholinergic neurons, dopanergic neurons, serotenergicneurons, and peptidergic neurons.

[0041] The present method is applicable, for example, to cell culturetechnique, such as in the culturing of neural and other cells whosesurvival or differentiative state is dependent on hedgehog function.Moreover, hedgehog agonists and antagonists can be used for therapeuticintervention, such as to enhance survival and maintenance of neurons andother neural cells in both the central nervous system and the peripheralnervous system, as well as to influence other vertebrate organogenicpathways, such as other ectodermal patterning, as well as certainmesodermal and endodermal differentiation processes. In an exemplaryembodiment, the method is practiced for modulating, in an animal, cellgrowth, cell differentiation or cell survival, and comprisesadministering a therapeutically effective amount of a hedgehogpolypeptide to alter, relative the absence of hedgehog treatment, atleast one of (i) rate of growth, (ii) differentiation, or (iii) survivalof one or more cell-types in the animal.

[0042] Another aspect of the present invention provides a method ofdetermining if a subject, e.g. a human patient, is at risk for adisorder characterized by unwanted cell proliferation or aberrantcontrol of differentiation. The method includes detecting, in a tissueof the subject, the presence or absence of a genetic lesioncharacterized by at least one of (i) a mutation of a gene encoding ahedgehog protein, e.g. represented in SEQ ID No: 2, or a homologthereof; or (ii) the mis-expression of a hedgehog gene. In preferredembodiments, detecting the genetic lesion includes ascertaining theexistence of at least one of: a deletion of one or more nucleotides froma hedgehog gene; an addition of one or more nucleotides to the gene, asubstitution of one or more nucleotides of the gene, a gross chromosomalrearrangement of the gene; an alteration in the level of a messenger RNAtranscript of the gene; the presence of a non-wild type splicing patternof a messenger RNA transcript of the gene; or a non-wild type level ofthe protein.

[0043] For example, detecting the genetic lesion can include (i)providing a probe/primer including an oligonucleotide containing aregion of nucleotide sequence which hybridizes to a sense or antisensesequence of a hedgehog gene, e.g. a nucleic acid represented in one ofSEQ ID Nos: 1-7, or naturally occurring mutants thereof, or 5′ or 3′flanking sequences naturally associated with the hedgehog gene; (ii)exposing the probe/primer to nucleic acid of the tissue; and (iii)detecting, by hybridization of the probe/primer to the nucleic acid, thepresence or absence of the genetic lesion; e.g. wherein detecting thelesion comprises utilizing the probe/primer to determine the nucleotidesequence of the hedgehog gene and, optionally, of the flanking nucleicacid sequences. For instance, the probe/primer can be employed in apolymerase chain reaction (PCR) or in a ligation chain reaction (LCR).In alternate embodiments, the level of a hedgehog protein is detected inan immunoassay using an antibody which is specifically immunoreactivewith the hedgehog protein.

[0044] The practice of the present invention will employ, unlessotherwise indicated, conventional techniques of cell biology, cellculture, molecular biology, transgenic biology, microbiology,recombinant DNA, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature. See, for example,Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritschand Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning,Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M.J. Gait ed., 1984); Mullis et al. U.S. Pat. No: 4,683,195; Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription AndTranslation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of AnimalCells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells AndEnzymes (IRL Press, 1986); B. Perbal, A Practical Guide To MolecularCloning (1984); the treatise, Methods In Enzymology (Academic Press,Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller andM. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods InEnzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical MethodsIn Cell And Molecular Biology (Mayer and Walker, eds., Academic Press,London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M.Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo,(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

[0045] Other features and advantages of the invention will be apparentfrom the following detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0046]FIG. 1 represents the amino acid sequences of two chick hh clones,chicken hedgehog-A (pCHA; SEQ ID No:35) and chicken hedgehog-B (pCHB;SEQ ID No:36). These clones were obtained using degenerate primerscorresponding to the underlined amino acid residues of the Drosophilasequence (corresponding to residues 161-232 of SEQ ID No:34) also shownin FIG. 1, followed by nested PCR using chicken genomic DNA.

[0047]FIG. 2 is an alignment comparing the amino acid sequences of chickShh (SEQ ID No:8) with its Drosophila homolog (SEQ ID No:34). Shhresidues 1-26 correspond to the proposed signal peptide. Identicalresidues are enclosed by boxes and gaps in order to highlightsimilarity. The nucleotide sequence of Shh has been submitted toGenbank.

[0048]FIG. 3 is a hydropathy plot for the predicted chick Shh protein,generated by the methods of Kyte and Doolittle (1982). The values ofhydrophobicity are plotted against the amino acid positions. Negativevalues predict a hydrophobic domain of the protein.

[0049]FIG. 4 is an alignment comparing the amino acid sequences ofvarious hh proteins. The white region on the amino terminus of chickenShh corresponds to the putative signal peptide. The black box refers toa highly conserved region from aa residues 26-207 of SEQ ID No:8). Thearrows point to exon boundaries in the Drosophila gene (Lee et al.(1992) Cell 71: 33-50). In each case, the proteins are compared tochicken Shh (SEQ ID No:8) and the percent amino acid identity isindicated in each region's box.

[0050]FIG. 5A is a “pileup” alignment of predicted amino acid sequenceswhich compares Drosophila hh (D-hh; SEQ ID No:34), mouse hh (M-Dhh; SEQID No:9; M-Ihh; SEQ ID No:10; M-Shh; SEQ ID No:11), chicken hh (C-Shh;SEQ ID No:8), and zebrafish hh (Z-Shh; SEQ ID No:12). The predictedhydrophobic transmembrane/signal sequences are indicated in italics andthe predicted signal sequence processing site is arrowed. The positionsof introns interrupting the Drosophila hh and M-Dhh open reading framesare indicated by arrowheads. All amino acids shared among the sixpredicted hh proteins are indicated in bold. FIG. 5B is a sequencealignment of the N-terminal portion of vertebrate hedgehog proteins, andthe predicted degenerate sequence “CON” (SEQ ID No: 41).

[0051]FIG. 6 is an inter- and cross-species comparison of amino acididentities among the predicted processed hh proteins shown in FIG. 5A.All values are percentages. Figures in parentheses representsimilarities allowing for conservative amino acid substitutions.

[0052]FIG. 7 is a representation of the DNA constructs used intransgenic studies to study ectopic expression of chick Shh in mouseembryos. Constructs were generated for ectopic expression of cDNA clonesin the Wnt-l expression domain and tested in transgenic mice embryosusing a lac-Z reporter (pWEXP-lacZ (used as a control)) and a chick Shhreporter (pWEXP-CShh). The pWEXP-CShh construct contained two tandemhead to tail copies of a chick Shh cDNA. The results of WEXP2-CShhtransgenic studies are shown in Table 1.

[0053]FIG. 8 is a model for anterioposterior limb patterning and theZone of Polarizing Activity (ZPA), based on Saunders and Gasseling(1968). The left portion of the diagram schematizes a stage 20 limb bud.The somites are illustrated as blocks along the left margin of the limbbud; right portion of the same panel illustrates the mature wing. Thehatched region on the posterior limb is the ZPA. Normally, the developedwing contains three digits II, III, and IV. The figure further shows theresult of transplanting a ZPA from one limb bud to the anterior marginof another. The mature limb now contains six digits IV, III, II, II,III, and IV in a mirror-image duplication of the normal pattern. Thelarge arrows in both panels represent the signal produced by the ZPAwhich acts to specify digit identity.

[0054]FIGS. 9A and 9B illustrate the comparison of zebrafish Shh (Z-Shh)and Drosophila hh (hh) amino acid sequences. FIG. 9A is an alignment ofzebrafish Shh and Drosophila hh amino acid sequences. Identical aminoacids are linked by vertical bars. Dots indicate gaps introduced foroptimal alignment. Putative transmembrane/signal peptide sequences areunderlined (Kyte and Doolittle (1982) J Mol Biol 157:133-148). Theposition of exon boundaries in the Drosophila gene are indicated byarrowheads. The region of highest similarity between Z-Shh and hhoverlaps exon 2. FIG. 9B is a schematic comparison of Z-Shh anddrosophila hh. Black boxes indicate the position of the putativetransmembrane/signal peptide sequences. relative to the amino-terminus.Sequence homologies were scored by taking into account the alignment ofchemically similar amino acids and percentage of homology in the boxedregions is indicated.

[0055]FIG. 10 is an alignment of partial predicted amino acid sequencesfrom three different zebrafish hh homologs. One of these sequencescorresponds to Shh, while the other two define additional hh homologs inzebrafish, named hh(a) and hh(b). Amino acid identities among the threepartial homologs are indicated by vertical bars.

[0056]FIG. 11 is a schematic representations of chick and mouse Shhproteins. The putative signal peptides and Asn-linked glycosylationsites are shown. The numbers refer to amino acid positions.

[0057]FIG. 12 is a schematic representation of myc-tagged Shhconstructs. The positions of the c-myc epitope tags are shown, as is thepredicted position of the proteolytic cleavage site. The shaded areafollowing the signal peptide of the carboxy terminal tagged constructrepresents the region included in the Glutathione-S-transferase fusionprotein used to generate antisera in rabbits.

[0058]FIG. 13 is a schematic diagram of Shh processing. Illustrated arecleavage of the signal peptide (black box), glycosylation at thepredicted Asn residue (N), and the secondary proteolytic cleavage. Thequestion marks indicate that the precise site of proteolytic cleavagehas not been determined. The different symbols representing thecarbohydrate moiety indicated maturation of this structure in the Golgiapparatus. The dashed arrow leading from the signal peptide cleavedprotein indicates that secretion of this species may be an artifact ofthe incomplete proteolytic processing of Shh seen in Xenopus oocytes andcos cells.

[0059]FIG. 14 is a schematic diagram of a model for the coordinatedgrowth and patterning of the limb. Sonic is proposed to signal directlyto the mesoderm to induce expression of the Hoxd and Bmp-2 genes. Theinduction of these mesodermal genes requires competence signals from theoverlying AER. One such signal is apparently Fgf-4. Expression of Fgf-4in the AER can be induced by Sonic providing an indirect signalingpathway from Sonic to the mesoderm. FGFs also maintain expression ofSonic in the ZPA, thereby completing a positive feedback loop whichcontrols the relative positions of the signaling centers. While Fgf-4provides competence signals to the mesoderm, it also promotes mesodermalproliferation. Thus patterning of the mesoderm is dependent on the samesignals which promote its proliferation. This mechanism inextricablyintegrates limb patterning with outgrowth.

[0060]FIG. 15 is a schematic diagram of patterning of the Drosophila andvertebrate gut. Regulatory interactions responsible for patterning ofDrosophila midgut (A) are compared to a model for patterning of thevertebrate hindgut (B) based on expression data. Morphologic regionaldistinctions are indicated to the left (A and B), genes expressed in thevisceral mesoderm are in the center panel, those in the gut lumenalendoderm are on the right. HOM/Hox gene expression domains are boxed.Regionally expressing secreted gene products are indicated by lines.Arrows indicate activating interactions, barred lines, inhibitinginteractions. Regulatory interactions in Drosophila gut (A) have beenestablished by genetic studies except for the relationship between dppand hedgehog, which is hypothesized based on their interactions in theDrosophila imaginal discs, hedgehog appears to be a signal from theendoderm to the mesoderm, and that dpp is expressed in the mesoderm.

[0061]FIG. 16 is a schematic diagram of chromosomal locations of Ihh,Shh and Dhh in the mouse genome. The loci were mapped by interspecificbackcross analysis. The segregation patterns of the loci and flankinggenes in backcross animals that were typed for all loci are shown abovethe chromosome maps. For individual pairs of loci more animals weretyped. Each column represents the chromosome identified in the backcrossprogeny that was inherited from the (C57BL/6J×M. spretus) F1 parent. Theshaded boxes represent the presence of a C57BL/6J allele and white boxesrepresent the presence of a M. spretus allele. The number of theoffsprings inheriting each type of chromosome is listed at the bottom ofeach column. Partial chromosome linkage maps showing location of Ihh,Shh and Dhh in relation too linked genes is shown. The number ofrecombinant N₂ animals is presented over totaFnumber of N₂ animals typedto the left of the chromosome maps between each pair of loci. Therecombinant frequencies, expressed as genetic distance in centimorgans(±one standard error) are also shown. When no recombination between lociwas detected, the upper 95% confidence limit of the recombinationdistance is indicated in parentheses. Gene order was determined byminimizing the number of recombinant events required to explain theallele distribution patterns. The position of loci in human chromosomescan be obtained from GDB (Genome Data Base), a computerized database ofhuman linkage information maintained by the William H. Welch MedicalLibrary of the John Hopkins University (Baltimore, Md.).

DETAILED DESCRIPTION OF THE INVENTION

[0062] Of particular importance in the development and maintenance oftissue in vertebrate animals is a type of extracellular communicationcalled induction, which occurs between neighboring cell layers andtissues (Saxen et al. (1989) Int J Dev Biol 33:21-48; and Gurdon et al.(1987) Development 99:285-306). In inductive interactions, chemicalsignals secreted by one cell population influence the developmental fateof a second cell population. Typically, cells responding to theinductive signals are diverted from one cell fate to another, neither ofwhich is the same as the fate of the signaling cells.

[0063] Inductive signals are key regulatory proteins that function invertebrate pattern formation, and are present in important signalingcenters known to operatex embryonically, for example, to define theorganization of the vertebrate embryo. For example, these signalingstructures include the notochord, a transient structure which initiatesthe formation of the nervous system and helps to define the differenttypes of neurons within it. The notochord also regulates mesodermalpatterning along the body axis. Another distinct group of cells havingapparent signaling activity is the floorplate of the neural tube (theprecursor of the spinal cord and brain) which also signals thedifferentiation of different nerve cell types. It is also generallybelieved that the region of mesoderm at the bottom of the buds whichform the limbs (called the Zone of Polarizing Activity or ZPA) operatesas a signaling center by secreting a morphogen which ultimately producesthe correct patterning of the developing limbs.

[0064] The present invention concerns the discovery that polypeptidesencoded by a family of vertebrate genes, termed here hedgehog genes,comprise the signals produced by these embryonic patterning centers. Asdescribed herein, each of the disclosed vertebrate hedgehog (hh)homologs exhibits spatially and temporally restricted expression domainsindicative of important roles in embryonic patterning. For instance, theresults provided below indicate that vertebrate hh genes are expressedin the posterior limb bud, Hensen's node, the early notochord, the floorplate of the neural tube, the fore- and hindgut and their derivatives.These are all important signaling centers known to be required forproper patterning of surrounding embryonic tissues.

[0065] The hedgehog family of vertebrate inter-cellular signalingmolecules provided by the present invention consists of at least fourmembers. Three of these members, herein referred to as Desert hedgehog(Dhh), Sonic hedgehog (Shh) and Indian hedgehog (Jhh), apparently existin all vertebrates, including fish, birds, and mammals. A fourth member,herein referred to as Moonrat hedgehog (Mhh), appears specific to fish.According to the appended sequence listing, (see also Table 1) a chickenShh polypeptide is encoded by SEQ ID No:1; a mouse Dhh polypeptide isencoded by SEQ ID No:2; a mouse Ihh polypeptide is encoded by SEQ IDNo:3; a mouse Shh polypeptide is encoded by SEQ ID No:4 a zebrafish Shhpolypeptide is encoded by SEQ ID No:5; a human Shh polypeptide isencoded by SEQ ID No:6; and a human Ihh polypeptide is encoded by SEQ IDNo:7. TABLE 1 Guide to hedgehog sequences in Sequence Listing NucleotideAmino Acid Chicken Shh SEQ ID No. 1 SEQ ID No. 8 Mouse Dhh SEQ ID No. 2SEQ ID No. 9 Mouse Ihh SEQ ID No. 3 SEQ ID No. 10 Mouse Shh SEQ ID No. 4SEQ ID No. 11 Zebrafish Shh SEQ ID No. 5 SEQ ID No. 12 Human Shh SEQ IDNo. 6 SEQ ID No. 13 Human Ihh SEQ ID No. 7 SEQ ID No. 14

[0066] Certain of the vertebrate hedgehog (hh) proteins of the presentinvention are defined by SEQ ID Nos:8-14 and can be cloned fromvertebrate organisms including fish, avian and mammalian sources. Theseproteins are distinct from the drosophila hedgehog protein which, forclarity, will be referred to hereinafter as “Dros-HH”. In addition tothe sequence variation between the various hh homologs, the vertebratehedgehog proteins are apparently present naturally in a number ofdifferent forms, including a pro-form, a full-length mature form, andseveral processed fragments thereof. The pro-form includes an N-terminalsignal peptide for directed secretion of the extracellular domain, whilethe fill-length mature form lacks this signal sequence. Furtherprocessing of the mature form apparently occurs in some instances toyield biologically active fragments of the protein. For instance, sonichedgehog undergoes additional proteolytic processing to yield twopeptides of approximately 19 kDa and 27 kDa, both of which are secreted.In addition to proteolytic fragmentation, the vertebrate hedgehogproteins can also be modified post-translationally, such as byglycosylation, though bacterially produced (e.g. unglycosylated) formsof the proteins apparently still maintain some of the activity of thenative protein.

[0067] As described in the following examples, the cDNA clones providedby the present invention were first obtained by screening a mousegenomic library with a partial Drosophila hh cDNA clone (0.7 kb).Positive plaques were identified and one mouse clone was selected. Thisclone was then used as a probe to obtain a genomic clone containing thefull coding sequence of the Mouse Dhh gene. As described in the attachedExamples, Northern blots and in situ hybridization demonstrated thatMouse Dhh is expressed in the testes, and potentially the ovaries, andis also associated with sensory neurons of the head and trunk.Interestingly, no expression was detected on the nerve cell bodiesthemselves (only the axons), indicating that Dhh is likely produced bythe Shwann cells.

[0068] In order to obtain cDNA clones encoding chicken hh genes,degenerate oligonucleotides were designed corresponding to the amino andcarboxy ends of Drosophila hh exon 2. As described in the Examplesbelow, these oligonucleotides were used to isolate PCR fragments fromchicken genomic DNA. These fragments were then cloned and sequenced. Tenclones yielded two different hh homologs, chicken Dhh and chicken Shh.The chicken Shh clone was then used to screen a stage 21/22 limb budcDNA library which yielded a full length Shh clone.

[0069] In order to identify other vertebrate hedgehog homologs, thechicken clones (Dhh and Shh) were used to probe a genomic southern blotcontaining chicken DNA. As described below, genomic DNA was cut withvarious enzymes which do not cleave within the probe sequences. The DNAwas run on a gel and transferred to a nylon filter. Probes were derivedby ligating each 220 bp clone into a concatomer and then labeling with arandom primer kit. The blots were hybridized and washed at lowstringency. In each case, three hybridizing bands were observedfollowing autoradiography, one of which was significantly more intense(a different band with each probe), indicating that there are at leastthree vertebrate hh genes. Additional cDNA and genomic screens carriedout have yielded clones of three hh homologs from chickens and mice(Shh, Dhh and Ihh), and four hh homologs from zebrafish (Shh, Dhh, Ihhand Mhh). Weaker hybridization signals suggested that the gene familymay be even larger. Moreover, a number of weakly hybridizing genomicclones have been isolated. Subsequently, the same probes derived fromchicken hedgehog homologs have been utilized to screen a human genomiclibrary. PCR fragments derived from the human genomic library were thensequenced, and PCR probes derived from the human sequences were used toscreen human fetal cDNA libraries. Full-length cDNA encoding human sonichedgehog protein (Shh) and partial cDNA encoding human Indian hedgehogprotein (Ihh) were isolated from the fetal library, and represent asource of recombinant human hedgehog proteins.

[0070] To order to determine the expression patterns of the variousvertebrate hh homologs, in situ hybridizations were performed indeveloping embryos of chicken, mice and fish. As described in theExamples below, the resulting expression patterns of each hh homologwere similar across each species and revealed that hh genes areexpressed in a number of important embryonic signaling centers. Forexample, Shh is expressed in Hensen's node, the notochord, the ventralfloorplate of the developing neural tube, and the ZPA at the base of thelimb buds; Ihh is expressed in the embryonic yolksac and hindgut, andappear also to be involved in chondrogenesis; Dhh is expressed in thetestes; and Mhh (only in zebrafish) is expressed in the notochord and incertain cranial nerves.

[0071] Furthermore, experimental evidence indicates that certainhedgehog proteins initiate expression of secondary signaling molecules,including Bmp-2 (a TGF-β relative) in the mesoderm and Fgf-4 in theectoderm. The mesoderm requires ectodermally-derived competencefactor(s), which include Fgf-4, to activate target gene expression inresponse to hedgehog signaling. The expression of, for example, Sonicand Fgf-4 is coordinately regulated by a positive feedback loopoperating between the posterior mesoderm and the overlying AER, which isthe ridge of pseudostratified epithelium extending antero-posteriorlyalong the distal margin of the bud. These data provide a basis forunderstanding the integration of growth and patterning in the developinglimb which can have important implications in the treatment of bonedisorders described in greater detail herein.

[0072] To determine the role hedgehog proteins plays in inductiveinteractions between the endoderm and mesoderm, which are critical togut morphogenesis, in situ hybridizations and recombinant retroviralinjections were performed in developing chick embryos. The ventralmesoderm is induced to undergo gut-specific differentiation by theadjacent endoderm. As described in Examples below, at the earlieststages of chick gut formation Shh is expressed by the endoderm, andBMP-4 (a TGF-β relative) is expressed in the adjacent visceral mesoderm.Ectopic expression of Sonic is sufficient to induce expression of BMP-4in visceral mesoderm, suggesting that Sonic serves as an inductivesignal from the endoderm to the mesoderm. Subsequent organ-specificendodermal differentiation depends on regional inductive signal from thevisceral mesoderm. Hox genes are expressed in the undifferentiated chickhind gut mesoderm with boundaries corresponding to morphologic borders,suggesting a role in regulating gut morphogenesis.

[0073] Bioactive fragments of hedgehog polypeptides of the presentinvention have been generated and are described in great detail in U.S.Ser. No. 08/435,093, filed May 4, 1995, herein incorporated byreference.

[0074] Accordingly, certain aspects of the present invention relate tonucleic acids encoding vertebrate hedgehog proteins, the hedgehogproteins themselves, antibodies immunoreactive with hh proteins, andpreparations of such compositions. Moreover, the present inventionprovides diagnostic and therapeutic assays and reagents for detectingand treating disorders involving, for example, aberrant expression ofvertebrate hedgehog homologs. In addition, drug discovery assays areprovided for identifying agents which can modulate the binding ofvertebrate hedgehog homologues to hedgehog-binding moieties (such ashedgehog receptors, ligands, or other extracellular matrix components).Such agents can be useful therapeutically to alter the growth and/ordifferentiation of a cell. Other aspects of the invention are describedbelow or will be apparent to those skilled in the art in light of thepresent disclosure.

[0075] For convenience, certain terms employed in the specification,examples, and appended claims are collected here.

[0076] As used herein, the term “nucleic acid” refers to polynucleotidessuch as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleicacid (RNA). The term should also be understood to include, asequivalents, analogs of either RNA or DNA made from nucleotide analogs,and, as applicable to the embodiment being described, single (sense orantisense) and double-stranded polynucleotides.

[0077] As used herein, the term “gene” or “recombinant gene” refers to anucleic acid comprising an open reading frame encoding one of thevertebrate hh polypeptides of the present invention, including both exonand (optionally) intron sequences. A “recombinant gene” refers tonucleic acid encoding a vertebrate hh polypeptide and comprisingvertebrate hh-encoding exon sequences, though it may optionally includeintron sequences which are either derived from a chromosomal vertebratehh gene or from an unrelated chromosomal gene. Exemplary recombinantgenes encoding the subject vertebrate hh polypeptide are represented bySEQ ID No:1, SEQ ID No:2, SEQ ID No:3, SEQ ID No:4, SEQ ID No:5, SEQ IDNo:6 or SEQ ID No:7. The term “intron” refers to a DNA sequence presentin a given vertebrate hh gene which is not translated into protein andis generally found between exons.

[0078] As used herein, the term “transfection” means the introduction ofa nucleic acid, e.g., an expression vector, into a recipient cell bynucleic acid-mediated gene transfer. “Transformation”, as used herein,refers to a process in which a cell's genotype is changed as a result ofthe cellular uptake of exogenous DNA or RNA, and, for example, thetransformed cell expresses a recombinant form of a vertebrate hhpolypeptide or, where anti-sense expression occurs from the transferredgene, the expression of a naturally-occurring form of the vertebrate hhprotein is disrupted.

[0079] As used herein the term “bioactive fragment of a hedgehogprotein” refers to a fragment of a hedgehog polypeptide, wherein theencoded polypeptide specifically agonizes or antagonizes inductiveevents mediated by wild-type hedgehog proteins. The hedgehog biactivefragment preferably is, for example, at least 5, 10, 20, 50, 100, 150 or200 amino acids in length.

[0080] An “effective amount” of a hedgehog polypeptide, or a bioactivefragment thereof, with respect to the subject method of treatment,refers to an amount of agonist or antagonist in a preparation which,when applied as part of a desired dosage regimen, provides modulation ofgrowth, differentiation or survival of cells, e.g., modulation ofspermatogenesis, skeletogenesis, e.g., osteogenesis, chondrogenesis, orlimb patterning, or neuronal differentiation.

[0081] As used herein, “phenotype” refers to the entire physical,biochemical, and physiological makeup of a cell, e.g., having any onetrait or any group of traits.

[0082] The terms “induction” or “induce”, as relating to the biologicalactivity of a hedgehog protein, refers generally to the process or actof causing to occur a specific effect on the phenotype of cell. Sucheffect can be in the form of causing a change in the phenotype, e.g.,differentiation to another cell phenotype, or can be in the form ofmaintaining the cell in a particular cell, e.g., preventingdedifferentation or promoting survival of a cell.

[0083] As used herein the term “animal” refers to mammals, preferablymammals such as live stock or humans. Likewise, a “patient” or “subject”to be treated by the subject method can mean either a human or non-humananimal.

[0084] As used herein, the term “vector” refers to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked. One type of preferred vector is an episome, i.e., a nucleicacid capable of extra-chromosomal replication. Preferred vectors arethose capable of autonomous replication and/expression of nucleic acidsto which they are linked. Vectors capable of directing the expression ofgenes to which they are operatively linked are referred to herein as“expression vectors”. In general, expression vectors of utility inrecombinant DNA techniques are often in the form of “plasmids” whichrefer generally to circular double stranded DNA loops which, in theirvector form are not bound to the chromosome. In the presentspecification, “plasmid” and “vector” are used interchangeably as theplasmid is the most commonly used form of vector. However, the inventionis intended to include such other forms of expression vectors whichserve equivalent functions and which become known in the artsubsequently hereto.

[0085] “Transcriptional regulatory sequence” is a generic term usedthroughout the specification to refer to DNA sequences, such asinitiation signals, enhancers, and promoters, which induce or controltranscription of protein coding sequences with which they are operablylinked. In preferred embodiments, transcription of one of therecombinant vertebrate hedgehog genes is under the control of a promotersequence (or other transcriptional regulatory sequence) which controlsthe expression of the recombinant gene in a cell-type in whichexpression is intended. It will also be understood that the recombinantgene can be under the control of transcriptional regulatory sequenceswhich are the same or which are different from those sequences whichcontrol transcription of the naturally-occurring forms of hedgehogproteins.

[0086] As used herein, the term “tissue-specific promoter” means a DNAsequence that serves as a promoter, i.e., regulates expression of aselected DNA sequence operably linked to the promoter, and which effectsexpression of the selected DNA sequence in specific cells of a tissue,such as cells of neural origin, e.g. neuronal cells. The term alsocovers so-called “leaky” promoters, which regulate expression of aselected DNA primarily in one tissue, but cause expression in othertissues as well.

[0087] As used herein, the term “target tissue” refers to connectivetissue, cartilage, bone tissue or limb tissue, which is either presentin an animal, e.g., a mammal, e.g., a human or is present in in vitroculture, e.g, a cell culture.

[0088] As used herein, a “transgenic animal” is any animal, preferably anon-human mammal, bird or an amphibian, in which one or more of thecells of the animal contain heterologous nucleic acid introduced by wayof human intervention, such as by transgenic techniques well known inthe art. The nucleic acid is introduced into the cell, directly orindirectly by introduction into a precursor of the cell, by way ofdeliberate genetic manipulation, such as by microinjection or byinfection with a recombinant virus. The term genetic manipulation doesnot include classical cross-breeding, or in vitro fertilization, butrather is directed to the introduction of a recombinant DNA molecule.This molecule may be integrated within a chromosome, or it may beextrachromosomally replicating DNA. In the typical transgenic animalsdescribed herein, the transgene causes cells to express a recombinantform of one of the vertebrate hh proteins, e.g. either agonistic orantagonistic forms. However, transgenic animals in which the recombinantvertebrate hh gene is silent are also contemplated, as for example, theFLP or CRE recombinase dependent constructs described below. The“non-human animals” of the invention include vertebrates such asrodents, non-human primates, sheep, dog, cow, chickens, amphibians,reptiles, etc. Preferred non-human animals are selected from the rodentfamily including rat and mouse, most preferably mouse, though transgenicamphibians, such as members of the Xenopus genus, and transgenicchickens can also provide important tools for understanding andidentifying agents which can affect, for example, embryogenesis andtissue formation. The term “chimeric animal” is used herein to refer toanimals in which the recombinant gene is found, or in which therecombinant is expressed in some but not all cells of the animal. Theterm “tissue-specific chimeric animal” indicates that one of therecombinant vertebrate hh genes is present and/or expressed in sometissues but not others.

[0089] As used herein, the term “transgene” means a nucleic acidsequence (encoding, e.g., one of the vertebrate hh polypeptides), whichis partly or entirely heterologous, i.e., foreign, to the transgenicanimal or cell into which it is introduced, or, is homologous to anendogenous gene of the transgenic animal or cell into which it isintroduced, but which is designed to be inserted, or is inserted, intothe animal's genome in such a way as to alter the genome of the cellinto which it is inserted (e.g., it is inserted at a location whichdiffers from that of the natural gene or its insertion results in aknockout). A transgene can include one or more transcriptionalregulatory sequences and any other nucleic acid, such as introns, thatmay be necessary for optimal expression of a selected nucleic acid.

[0090] As is well known, genes for a particular polypeptide may exist insingle or multiple copies within the genome of an individual. Suchduplicate genes may be identical or may have certain modifications,including nucleotide substitutions, additions or deletions, which allstill code for polypeptides having substantially the same activity. Theterm “DNA sequence encoding a vertebrate hh polypeptide” may thus referto one or more genes within a particular individual. Moreover, certaindifferences in nucleotide sequences may exist between individualorganisms, which are called alleles. Such allelic differences may or maynot result in differences in amino acid sequence of the encodedpolypeptide yet still encode a protein with the same biologicalactivity.

[0091] “Homology” refers to sequence similarity between two peptides orbetween two nucleic acid molecules. Homology can be determined bycomparing a position in each sequence which may be aligned for purposesof comparison. When a position in the compared sequence is occupied bythe same base or amino acid, then the molecules are homologous at thatposition. A degree of homology between sequences is a function of thenumber of matching or homologous positions shared by the sequences. An“unrelated” or “non-homologous” sequence shares less than 40 percentidentity, though preferably less than 25 percent identity, with one ofthe vertebrate hh sequences of the present invention.

[0092] “Cells,” “host cells” or “recombinant host cells” are terms usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but to the progeny or potential progenyof such a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

[0093] A “chimeric protein” or “fusion protein” is a fusion of a firstamino acid sequence encoding one of the subject vertebrate hhpolypeptides with a second amino acid sequence defining a domain foreignto and not substantially homologous with any domain of one of thevertebrate hh proteins. A chimeric protein may present a foreign domainwhich is found (albeit in a different protein) in an organism which alsoexpresses the first protein, or it may be an “interspecies”,“intergenic”, etc. fusion of protein structures expressed by differentkinds of organisms. In general, a fusion protein can be represented bythe general formula X-hh-Y, wherein hh represents a portion of theprotein which is derived from one of the vertebrate hh proteins, and Xand Y are independently absent or represent amino acid sequences whichare not related to one of the vertebrate hh sequences in an organism,including naturally occurring mutants.

[0094] As used herein, the terms “transforming growth factor-beta” and“TGF-β” denote a family of structurally related paracrine polypeptidesfound ubiquitously in vertebrates, and prototypic of a large family ofmetazoan growth, differentiation, and morphogenesis factors (see, forreview, Massaque et al. (1990) Ann Rev Cell Biol 6:597-641; and Sporn etal. (1992) J Cell Biol 119:1017-1021). Included in this family are the“bone morphogenetic proteins” or “BMPs”, which refers to proteinsisolated from bone, and fragments thereof and synthetic peptides whichare capable of inducing bone deposition alone or when combined withappropriate cofactors. Preparation of BMPs, such as BMP-1, -2, -3, and-4, is described in, for example, PCT publication WO 88/00205. Wozney(1989) Growth Fact Res 1:267-280 describes additional BMP proteinsclosely related to BMP-2, and which have been designated BMP-5, -6, and-7. PCT publications WO89/09787 and WO89/09788 describe a protein called“OP-1,” now known to be BMP-7. Other BMPs are known in the art.

[0095] The term “isolated” as also used herein with respect to nucleicacids, such as DNA or RNA, refers to molecules separated from otherDNAs, or RNAs, respectively, that are present in the natural source ofthe macromolecule. For example, an isolated nucleic acid encoding one ofthe subject vertebrate hh polypeptides preferably includes no more than10 kilobases (kb) of nucleic acid sequence which naturally immediatelyflanks the vertebrate hh gene in genomic DNA, more preferably no morethan 5 kb of such naturally occurring flanking sequences, and mostpreferably less than 1.5 kb of such naturally occurring flankingsequence. The term isolated as used herein also refers to a nucleic acidor peptide that is substantially free of cellular material, viralmaterial, or culture medium when produced by recombinant DNA techniques,or chemical precursors or other chemicals when chemically synthesized.Moreover, an “isolated nucleic acid” is meant to include nucleic acidfragments which are not naturally occurring as fragments and would notbe found in the natural state.

[0096] As used herein the term “approximately 19 kDa” with respect toN-terminal bioactive fragments of a hedgehog protein, refers to apolypeptide which can range in size from 16 kDa to 22 kDa, morepreferably 18-20 kDa. In a preferred embodiment, “approximately 19 kDa”refers to a mature form of the peptide after the cleavage of the signalsequence and proteolysis to release an N-terminal portion of the matureprotein. For instance, in the case of the Sonic hedgehog polypeptide, afragment of approximately 19 kDa is generated when the maturepolypeptide is cleaved at a proteolytic processing site which is locatedin the region between Ala-169 and Gly-178 of SEQ ID No:40, e.g., afragment from Cys-1 to Gly-174 of SEQ ID No:40.

[0097] Likewise, the term “approximately 27 kDa” with respect toC-terminal fragments of a hedgehog protein, refers to a polypeptidewhich can range in size from 24 kDa to 30 kDa, more preferably 26-29kDa. In a preferred embodiment, “approximately 27 kDa” refers to amature form of the C-terminal polypeptide after proteolysis to releasean N-terminal portion of the mature protein.

[0098] As described below, one aspect of the invention pertains toisolated nucleic acids comprising the nucleotide sequences encodingvertebrate hh homologues, and/or equivalents of such nucleic acids. Theterm nucleic acid as used herein is intended to include fragments asequivalents. The term equivalent is understood to include nucleotidesequences encoding functionally equivalent hedgehog polypeptides orfunctionally equivalent peptides having an activity of a vertebratehedgehog protein such as described herein. Equivalent nucleotidesequences will include sequences that differ by one or more nucleotidesubstitutions, additions or deletions, such as allelic variants; andwill, therefore, include sequences that differ from the nucleotidesequence of the vertebrate hedgehog cDNAs shown in SEQ ID Nos:1-7 due tothe degeneracy of the genetic code. Equivalents will also includenucleotide sequences that hybridize under stringent conditions (i.e.,equivalent to about 20-27° C. below the melting temperature (T_(m)) ofthe DNA duplex formed in about 1M salt) to the nucleotide sequencesrepresented in one or more of SEQ ID Nos:1-7. In one embodiment,equivalents will further include nucleic acid sequences derived from andevolutionarily related to, a nucleotide sequences shown in any of SEQ IDNos:1-7.

[0099] Moreover, it will be generally appreciated that, under certaincircumstances, it may be advantageous to provide homologs of one of thesubject hedgehog polypeptides which function in a limited capacity asone of either a hedgehog agonist (mimetic) or a hedgehog antagonist, inorder to promote or inhibit only a subset of the biological activitiesof the naturally-occurring form of the protein. Thus, specificbiological effects can be elicited by treatment with a homolog oflimited function, and with fewer side effects relative to treatment withagonists or antagonists which are directed to all of the biologicalactivities of naturally occurring forms of hedgehog proteins.

[0100] Homologs of one of the subject hedgehog proteins can be generatedby mutagenesis, such as by discrete point mutation(s), or by truncation.For instance, mutation can give rise to homologs which retainsubstantially the same, or merely a subset, of the biological activityof the hh polypeptide from which it was derived. Alternatively,antagonistic forms of the protein can be generated which are able toinhibit the function of the naturally occurring form of the protein,such as by competitively binding to an hh receptor.

[0101] In general, polypeptides referred to herein as having an activity(e.g., are “bioactive”) of a vertebrate hh protein are defined aspolypeptides which include an amino acid sequence corresponding (e.g.,identical or homologous) to all or a portion of the amino acid sequencesof a vertebrate hh proteins shown in any of SEQ ID No:8, SEQ ID No:9,SEQ ID No:10, SEQ ID No:11, SEQ ID No:12, SEQ ID No:13 or SEQ ID No:14and which mimic or antagonize all or a portion of thebiological/biochemical activities of a naturally occuring hedgehogprotein. Examples of such biological activity include the ability toinduce (or otherwise modulate) formation and differentiation of thehead, limbs, lungs, central nervous system (CNS), digestive tract orother gut components, or mesodermal patterning of developing vertebrateembryos. As set out in U.S. Ser. Nos. 08/356,060 and 08/176,427, thevertebrate hedgehog proteins, especially Shh, can constitute a generalventralizing activity. For instance, the subject polypeptides can becharacterized by an ability to induce and/or maintain differentiation ofneurons, e.g., motorneurons, cholinergic neurons, dopanergic neurons,serotenergic neurons, peptidergic neurons and the like. In preferredembodiments, the biological activity can comprise an ability to regulateneurogenesis, such as a motor neuron inducing activity, a neuronaldifferentiation inducing activity, or a neuronal survival promotingactivity. Hedgehog proteins of the present invention can also havebiological activities which include an ability to regulate organogensis,such as through the ability to influence limb patterning, by, forexample, skeletogenic activity. The biological activity associated withthe hedgehog proteins of the present invention can also include theability to induce stem cell or germ cell differentiation, including theability to induce differentiation of chondrocytes or an involvement inspermatogenesis.

[0102] Hedgehog proteins of the present invention can also becharacterized in terms of biological activities which include: anability to modulate proliferation, survival and/or differentiation ofmesodermally-derived tissue, such as tissue derived from dorsalmesoderm; the ability to modulate proliferation, survival and/ordifferentiation of ectodermally-derived tissue, such as tissue derivedfrom the neural tube, neural crest, or head mesenchyme; the ability tomodulate proliferation, survival and/or differentiation ofendodermally-derived tissue, such as tissue derived from the primitivegut. Moreover, as described in the Examples below, the subject hedgehogproteins have the ability to induce expression of secondary signalingmolecules, such as members of the Transforming Growth Factor β (TGFβ)family, including bone morphogenic proteins, e.g. BMP-2 and BMP-4, aswell as members of the fibroblast growth factor (FGF) family, such asFgf-4. Other biological activities of the subject hedgehog proteins aredescribed herein or will be reasonably apparent to those skilled in theart. According to the present invention, a polypeptide has biologicalactivity if it is a specific agonist or antagonist of anaturally-occurring form of a vertebrate hedgehog protein.

[0103] Preferred nucleic acids encode a vertebrate hedgehog polypeptidecomprising an amino acid sequence at least 60% homologous, morepreferably 70% homologous and most preferably 80% homologous with anamino acid sequence selected from the group consisting of SEQ IDNos:8-14. Nucleic acids which encode polypeptides at least about 90%,more preferably at least about 95%, and most preferably at least about98-99% homology with an amino acid sequence represented in one of SEQ IDNos:8-14 are also within the scope of the invention. In one embodiment,the nucleic acid is a cDNA encoding a peptide having at least oneactivity of the subject vertebrate hh polypeptide. Preferably, thenucleic acid includes all or a portion of the nucleotide sequencecorresponding to the coding region of SEQ ID Nos:1-7.

[0104] Preferred nucleic acids encode a bioactive fragment of avertebrate hedgehog polypeptide comprising an amino acid sequence atleast 60% homologous, more preferably 70% homologous and most preferably80% homologous with an amino acid sequence selected from the groupconsisting of SEQ ID Nos:8-14. Nucleic acids which encode polypeptidesat least about 90%, more preferably at least about 95%, and mostpreferably at least about 98-99% homology, or identical, with an aminoacid sequence represented in one of SEQ ID Nos:8-14 are also within thescope of the invention.

[0105] With respect to biocvive fragments of sonic clones, a preferrednucleic acid encodes a polypeptide including a hedgehog portion havingmolecular weight of approximately 19 kDa and which polyptide canmodulate, e.g., mimic or antagonize, a hedgehog biological activity.Preferably, the polypeptide encoded by the nucleic acid comprises anamino acid sequence identical or homologous to an amino acid sequencedesignated in one of SEQ ID No:8, SEQ ID No:9, SEQ ID No:10, SEQ IDNo:11, SEQ ID No:12, SEQ ID No:13, or SEQ ID No:14. More preferably, thepolypeptide comprises an amino acid sequence designated in SEQ ID No:40.

[0106] A preferred nucleic acid encodes a hedgehog polypeptidecomprising an amino acid sequence represented by the formula A-Bwherein, A represents all or the portion of the amino acid sequencedesignated by residues 1-168 of SEQ ID No:40; and B represents at leastone amino acid residue of the amino acid sequence designated by residues169-221 of SEQ ID No:40; wherein A and B together represent a contiguouspolypeptide sequence designated by SEQ ID No:40. Preferably, B canrepresent at least five, ten or twenty amino acid residues of the aminoacid sequence designated by residues 169-221 of SEQ ID No:40.

[0107] To further illustrate, another preferred nucleic acid encodes apolypeptide comprising an amino acid sequence represented by the formulaA-B, wherein A represents all or the portion of the amino acid sequencedesignated by residues 24-193 of SEQ ID No:13; and B represents at leastone amino acid residue of the amino acid sequence designated by residues194-250 of SEQ ID No:13; wherein A and B together represent a contiguouspolypeptide sequence designated in SEQ ID No:13, and the polypeptidemodulates, e.g., agonizes or antagonizes, the biological activity of ahedgehog protein.

[0108] Yet another preferred nucleic acid encodes a polypeptidecomprising an amino acid sequence represented by the formula A-B,wherein A represents all or the portion, e.g., 25, 50, 75 or 100residues, of the amino acid sequence designated by residues 25-193, oranalogous residues thereof, of a vertebrate hedgehog polypeptideidentical or homologous to SEQ ID No:11; and B represents at least oneamino acid residue of the amino acid sequence designated by residues194-250, or analogous residues thereof, of a vertebrate hedgehogpolypeptide identical or homologous to SEQ ID No:11; wherein A and Btogether represent a contiguous polypeptide sequence designated in SEQID No:11.

[0109] Another preferred nucleic acid encodes a polypeptide comprisingan amino acid sequence represented by the formula A-B, wherein Arepresents all or the portion, e.g., 25, 50, 75 or 100 residues, of theamino acid sequence designated by residues 23-193 of SEQ ID No:9; and Brepresents at least one amino acid residue of the amino acid sequencedesignated by residues 194-250 of SEQ ID No:9; wherein A and B togetherrepresent a contiguous polypeptide sequence designated in SEQ ID No:9,and the polypeptide modulates, e.g., agonizes or antagonizes, thebiological activity of a hedgehog protein.

[0110] Another preferred nucleic acid encodes a polypeptide comprisingan amino acid sequence represented by the formula A-B, wherein Arepresents all or the portion, e.g., 25, 50, 75 or 100 residues, of theamino acid sequence designated by residues 28-197 of SEQ ID No:10; and Brepresents at least one amino acid residue of the amino acid sequencedesignated by residues 198-250 of SEQ ID No:10; wherein A and B togetherrepresent a contiguous polypeptide sequence designated in SEQ ID No:10,and the polypeptide modulates, e.g., agonizes or antagonizes, thebiological activity of a hedgehog protein.

[0111] Yet another preferred nucleic acid encodes a polypeptidecomprising an amino acid sequence represented by the formula A-B,wherein A represents all or the portion, e.g., 25, 50 or 75 residues, ofthe amino acid sequence designated by residues 1-98, or analogousresidues thereof, of a vertebrate hedgehog polypeptide identical orhomologous to SEQ ID No:14; and B represents at least one amino acidresidue of the amino acid sequence designated by residues 99-150, oranalogous residues thereof, of a vertebrate hedgehog polypeptideidentical or homologous to SEQ ID No:14; wherein A and B togetherrepresent a contiguous polypeptide sequence designated in SEQ ID No:14.

[0112] Another aspect of the invention provides a nucleic acid whichhybridizes under high or low stringency conditions to a nucleic acidrepresented by one of SEQ ID Nos:1-7. Appropriate stringency conditionswhich promote DNA hybridization, for example, 6.0×sodium chloride/sodiumcitrate (SSC) at about 45° C., followed by a wash of 2.0×SSC at 50° C.,are known to those skilled in the art or can be found in CurrentProtocols in Molecular Biology, John Wiley & Sons, N.Y. (1989),6.3.1-6.3.6. For example, the salt concentration in the wash step can beselected from a low stringency of about 2.0×SSC at 50° C. to a highstringency of about 0.2×SSC at 50° C. In addition, the temperature inthe wash step can be increased from low stringency conditions at roomtemperature, about 22° C., to high stringency conditions at about 65° C.

[0113] Nucleic acids, having a sequence that differs from the nucleotidesequences shown in one of SEQ ID No:1, SEQ ID No:2, SEQ ID No:3, SEQ IDNo:4, SEQ ID No:5, SEQ ID No:6 or SEQ ID No:7 due to degeneracy in thegenetic code are also within the scope of the invention. Such nucleicacids encode functionally equivalent peptides (i.e., a peptide having abiological activity of a vertebrate hh polypeptide) but differ insequence from the sequence shown in the sequence listing due todegeneracy in the genetic code. For example, a number of amino acids aredesignated by more than one triplet. Codons that specify the same aminoacid, or synonyms (for example, CAU and CAC each encode histidine) mayresult in “silent” mutations which do not affect the amino acid sequenceof a vertebrate hh polypeptide. However, it is expected that DNAsequence polymorphisms that do lead to changes in the amino acidsequences of the subject hh polypeptides will exist among vertebrates.One skilled in the art will appreciate that these variations in one ormore nucleotides (up to about 3-5% of the nucleotides) of the nucleicacids encoding polypeptides having an activity of a vertebrate hhpolypeptide may exist among individuals of a given species due tonatural allelic variation.

[0114] As used herein, a hedgehog gene fragment refers to a nucleic acidhaving fewer nucleotides than the nucleotide sequence encoding theentire mature form of a vertebrate hh protein yet which (preferably)encodes a polypeptide which retains some biological activity of the fulllength protein.

[0115] As indicated by the examples set out below, hedgehogprotein-encoding nucleic acids can be obtained from mRNA present in anyof a number of eukaryotic cells. It should also be possible to obtainnucleic acids encoding vertebrate hh polypeptides of the presentinvention from genomic DNA obtained from both adults and embryos. Forexample, a gene encoding a hh protein can be cloned from either a cDNAor a genomic library in accordance with protocols described herein, aswell as those generally known to persons skilled in the art. A cDNAencoding a hedgehog protein can be obtained by isolating total mRNA froma cell, e.g. a mammalian cell, e.g. a human cell, including embryoniccells. Double stranded cDNAs can then be prepared from the total mRNA,and subsequently inserted into a suitable plasmid or bacteriophagevector using any one of a number of known techniques. The gene encodinga vertebrate hh protein can also be cloned using established polymerasechain reaction techniques in accordance with the nucleotide sequenceinformation provided by the invention. The nucleic acid of the inventioncan be DNA or RNA. A preferred nucleic acid is a cDNA represented by asequence selected from the group consisting of SEQ ID Nos: 1-7.

[0116] Another aspect of the invention relates to the use of theisolated nucleic acid in “antisense” therapy. As used herein,“antisense” therapy refers to administration or in situ generation ofoligonucleotide probes or their derivatives which specificallyhybridizes (e.g. binds) under cellular conditions, with the cellularmRNA and/or genomic DNA encoding one or more of the subject hedgehogproteins so as to inhibit expression of that protein, e.g. by inhibitingtranscription and/or translation. The binding may be by conventionalbase pair complementarity, or, for example, in the case of binding toDNA duplexes, through specific interactions in the major groove of thedouble helix. In general, “antisense” therapy refers to the range oftechniques generally employed in the art, and includes any therapy whichrelies on specific binding to oligonucleotide sequences

[0117] An antisense construct of the present invention can be delivered,for example, as an expression plasmid which, when transcribed in thecell, produces RNA which is complementary to at least a unique portionof the cellular mRNA which encodes a vertebrate hh protein.Alternatively, the antisense construct is an oligonucleotide probe whichis generated ex vivo and which, when introduced into the cell causesinhibition of expression by hybridizing with the mRNA and/or genomicsequences of a vertebrate hh gene. Such oligonucleotide probes arepreferably modified oligonucleotide which are resistant to endogenousnucleases, e.g. exonucleases and/or endonucleases, and is thereforestable in vivo. Exemplary nucleic acid molecules for use as antisenseoligonucleotides are phosphoramidate, phosphothioate andmethylphosphonate analogs of DNA (see also U.S. Pat. Nos. 5,176,996;5,264,564; and 5,256,775). Additionally, general approaches toconstructing oligomers useful in antisense therapy have been reviewed,for example, by Van der Krol et al. (1988) Biotechniques 6:958-976; andStein et al. (1988) Cancer Res 48:2659-2668.

[0118] Accordingly, the modified oligomers of the invention are usefulin therapeutic, diagnostic, and research contexts. In therapeuticapplications, the oligomers are utilized in a manner appropriate forantisense therapy in general. For such therapy, the oligomers of theinvention can be formulated for a variety of loads of administration,including systemic and topical or localized administration. Techniquesand formulations generally may be found in Remmington's PharmaceuticalSciences, Meade Publishing Co., Easton, Pa. For systemic administration,injection is preferred, including intramuscular, intravenous,intraperitoneal, and subcutaneous for injection, the oligomers of theinvention can be formulated in liquid solutions, preferably inphysiologically compatible buffers such as Hank's solution or Ringer'ssolution. In addition, the oligomers may be formulated in solid form andredissolved or suspended immediately prior to use. Lyophilized forms arealso included.

[0119] Systemic administration can also be by transmucosal ortransdermal means, or the compounds can be administered orally. Fortransmucosal or transdermal administration, penetrants appropriate tothe barrier to be permeated are used in the formulation. Such penetrantsare generally known in the art, and include, for example, fortransmucosal administration bile salts and fusidic acid derivatives. Inaddition, detergents may be used to facilitate permeation. Transmucosaladministration may be through nasal sprays or using suppositories. Fororal administration, the oligomers are formulated into conventional oraladministration forms such as capsules, tablets, and tonics. For topicaladministration, the oligomers of the invention are formulated intoointments, salves, gels, or creams as generally known in the art.

[0120] In addition to use in therapy, the oligomers of the invention maybe used as diagnostic reagents to detect the presence or absence of thetarget DNA or RNA sequences to which they specifically bind. Suchdiagnostic tests are described in further detail below.

[0121] Likewise, the antisense constructs of the present invention, byantagonizing the normal biological activity of one of the hedgehogproteins, can be used in the manipulation of tissue, e.g. tissuedifferentiation, both in vivo and in ex vivo tissue cultures.

[0122] Also, the anti-sense techniques (e.g. microinjection of antisensemolecules, or transfection with plasmids whose transcripts areanti-sense with regard to an hh mRNA or gene sequence) can be used toinvestigate role of hh in developmental events, as well as the normalcellular function of hh in adult tissue. Such techniques can be utilizedin cell culture, but can also be used in the creation of transgenicanimals.

[0123] This invention also provides expression vectors containing anucleic acid encoding a vertebrate hh polypeptide, operably linked to atleast one transcriptional regulatory sequence. Operably linked isintended to mean that the nucleotide sequence is linked to a regulatorysequence in a manner which allows expression of the nucleotide sequence.Regulatory sequences are art-recognized and are selected to directexpression of the subject vertebrate hh proteins. Accordingly, the termtranscriptional regulatory sequence includes promoters, enhancers andother expression control elements. Such regulatory sequences aredescribed in Goeddel; Gene Expression Technology: Methods in Enzymology185, Academic Press, San Diego, Calif. (1990). For instance, any of awide variety of expression control sequences, sequences that control theexpression of a DNA sequence when operatively linked to it, may be usedin these vectors to express DNA sequences encoding vertebrate hhpolypeptides of this invention. Such useful expression controlsequences, include, for example, a viral LTR, such as the LTR of theMoloney murine leukemia virus, the early and late promoters of SV40,adenovirus or cytomegalovirus immediate early promoter, the lac system,the trp system, the TAC or TRC system, T7 promoter whose expression isdirected by T7 RNA polymerase, the major operator and promoter regionsof phage λ, the control regions for fd coat protein, the promoter for3-phosphoglycerate kinase or other glycolytic enzymes, the promoters ofacid phosphatase, e.g., Pho5, the promoters of the yeast α-matingfactors, the polyhedron promoter of the baculovirus system and othersequences known to control the expression of genes of prokaryotic oreukaryotic cells or their viruses, and various combinations thereof. Itshould be understood that the design of the expression vector may dependon such factors as the choice of the host cell to be transformed and/orthe type of protein desired to be expressed. Moreover, the vector's copynumber, the ability to control that copy number and the expression ofany other proteins encoded by the vector, such as antibiotic markers,should also be considered. In one embodiment, the expression vectorincludes a recombinant gene encoding a peptide having an agonisticactivity of a subject hedgehog polypeptide, or alternatively, encoding apeptide which is an antagonistic form of the hh protein. Such expressionvectors can be used to transfect cells and thereby produce polypeptides,including fusion proteins, encoded by nucleic acids as described herein.

[0124] Moreover, the gene constructs of the present invention can alsobe used as a part of a gene therapy protocol to deliver nucleic acidsencoding either an agonistic or antagonistic form of one of the subjectvertebrate hedgehog proteins. Thus, another aspect of the inventionfeatures expression vectors for in vivo or in vitro transfection andexpression of a vertebrate hh polypeptide in particular cell types so asto reconstitute the function of, or alternatively, abrogate the functionof hedgehog-induced signaling in a tissue in which thenaturally-occurring form of the protein is misexpressed; or to deliver aform of the protein which alters differentiation of tissue, or whichinhibits neoplastic transformation.

[0125] Expression constructs of the subject vertebrate hh polypeptide,and mutants thereof, may be administered in any biologically effectivecarrier, e.g. any formulation or composition capable of effectivelydelivering the recombinant gene to cells in vivo. Approaches includeinsertion of the subject gene in viral vectors including recombinantretroviruses, adenovirus, adeno-associated virus, and herpes simplexvirus-1, or recombinant bacterial or eukaryotic plasmids. Viral vectorstransfect cells directly; plasmid DNA can be delivered with the help of,for example, cationic liposomes (lipofectin) or derivatized (e.g.antibody conjugated), polylysine conjugates, gramacidin S, artificialviral envelopes or other such intracellular carriers, as well as directinjection of the gene construct or CaPO₄ precipitation carried out invivo. It will be appreciated that because transduction of appropriatetarget cells represents the critical first step in gene therapy, choiceof the particular gene delivery system will depend on such factors asthe phenotype of the intended target and the route of administration,e.g. locally or systemically. Furthermore, it will be recognized thatthe particular gene construct provided for in vivo transduction ofhedgehog expression are also useful for in vitro transduction of cells,such as for use in the ex vivo tissue culture systems described below.

[0126] A preferred approach for in vivo introduction of nucleic acidinto a cell is by use of a viral vector containing nucleic acid, e.g. acDNA, encoding the particular form of the hedgehog polypeptide desired.Infection of cells with a viral vector has the advantage that a largeproportion of the targeted cells can receive the nucleic acid.Additionally, molecules encoded within the viral vector, e.g., by a cDNAcontained in the viral vector, are expressed efficiently in cells whichhave taken up viral vector nucleic acid.

[0127] Retrovirus vectors and adeno-associated virus vectors aregenerally understood to be the recombinant gene delivery system ofchoice for the transfer of exogenous genes in vivo, particularly intohumans. These vectors provide efficient delivery of genes into cells,and the transferred nucleic acids are stably integrated into thechromosomal DNA of the host. A major prerequisite for the use ofretroviruses is to ensure the safety of their use, particularly withregard to the possibility of the spread of wild-type virus in the cellpopulation. The development of specialized cell lines (termed “packagingcells”) which produce only replication-defective retroviruses hasincreased the utility of retroviruses for gene therapy, and defectiveretroviruses are well characterized for use in gene transfer for genetherapy purposes (for a review see Miller, A. D. (1990) Blood 76:271).Thus, recombinant retrovirus can be constructed in which part of theretroviral coding sequence (gag, pol, env) has been replaced by nucleicacid encoding one of the subject proteins rendering the retrovirusreplication defective. The replication defective retrovirus is thenpackaged into virions which can be used to infect a target cell throughthe use of a helper virus by standard techniques. Protocols forproducing recombinant retroviruses and for infecting cells in vitro orin vivo with such viruses can be found in Current Protocols in MolecularBiology, Ausubel, F. M. et al. (eds.) Greene Publishing Associates,(1989), Sections 9.10-9.14 and other standard laboratory manuals.Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM whichare well known to those skilled in the art. Examples of suitablepackaging virus lines for preparing both ecotropic and amphotropicretroviral systems include ψCrip, ψCre, ψ2 and ψAm. Retroviruses havebeen used to introduce a variety of genes into many different celltypes, including neuronal cells, in vitro and/or in vivo (see forexample Eglitis, et al. (1985) Science 230:1395-1398; Danos and Mulligan(1988) Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al. (1988)Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al. (1990) Proc.Natl. Acad. Sci. USA 87:6141-6145; Huber et al. (1991) Proc. Natl. Acad.Sci. USA 88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; vanBeusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay etal. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl.Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J. Immunol.150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCTApplication WO 89/07136; PCT Application WO 89/02468; PCT Application WO89/05345; and PCT Application WO 92/07573).

[0128] Furthermore, it has been shown that it is possible to limit theinfection spectrum of retroviruses and consequently of retroviral-basedvectors, by modifying the viral packaging proteins on the surface of theviral particle (see, for example PCT publications WO93/25234 andWO94/06920). For instance, strategies for the modification of theinfection spectrum of retroviral vectors include: coupling antibodiesspecific for cell surface antigens to the viral env protein (Roux et al.(1989) PNAS 86:9079-9083; Julan et al. (1992) J. Gen Virol 73:3251-3255;and Goud et al. (1983) Virology 163:251-254); or coupling cell surfacereceptor ligands to the viral env proteins (Neda et al. (1991) J BiolChem 266:14143-14146). Coupling can be in the form of the chemicalcross-linking with a protein or other variety (e.g. lactose to convertthe env protein to an asialoglycoprotein), as well as by generatingfusion proteins (e.g. single-chain antibody/env fusion proteins). Thistechnique, while useful to limit or otherwise direct the infection tocertain tissue types, can also be used to convert an ecotropic vector into an amphotropic vector.

[0129] Moreover, use of retroviral gene delivery can be further enhancedby the use of tissue- or cell-specific transcriptional regulatorysequences which control expression of the hh gene of the retroviralvector.

[0130] Another viral gene delivery system useful in the presentinvention utilizes adenovirus-derived vectors. The genome of anadenovirus can be manipulated such that it encodes and expresses a geneproduct of interest but is inactivated in terms of its ability toreplicate in a normal lytic viral life cycle. See for example Berkner etal. (1988) BioTechniques 6:616; Rosenfeld et al. (1991) Science252:431-434; and Rosenfeld et al. (1992) Cell 68:143-155. Suitableadenoviral vectors derived from the adenovirus strain Ad type 5 dl324 orother strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are well known tothose skilled in the art. Recombinant adenoviruses can be advantageousin certain circumstances in that they can be used to infect a widevariety of cell types, including airway epithelium (Rosenfeld et al.(1992) cited supra), endothelial cells (Lemarchand et al. (1992) Proc.Natl. Acad. Sci. USA 89:6482-6486), hepatocytes (Herz and Gerard (1993)Proc. Natl. Acad. Sci. USA 90:2812-2816) and muscle cells (Quantin etal. (1992) Proc. Natl. Acad. Sci. USA 89:2581-2584). Furthermore, thevirus particle is relatively stable and amenable to purification andconcentration, and as above, can be modified so as to affect thespectrum of infectivity. Additionally, introduced adenoviral DNA (andforeign DNA contained therein) is not integrated into the genome of ahost cell but remains episomal, thereby avoiding potential problems thatcan occur as a result of insertional mutagenesis in situations whereintroduced DNA becomes integrated into the host genome (e.g., retroviralDNA). Moreover, the carrying capacity of the adenoviral genome forforeign DNA is large (up to 8 kilobases) relative to other gene deliveryvectors (Berkner et al. cited supra; Haj-Ahmand and Graham (1986) J.Virol. 57:267). Most replication-defective adenoviral vectors currentlyin use and therefore favored by the present invention are deleted forall or parts of the viral E1 and E3 genes but retain as much as 80% ofthe adenoviral genetic material (see, e.g., Jones et al. (1979) Cell16:683; Berkner et al., supra; and Graham et al. in Methods in MolecularBiology, E. J. Murray, Ed. (Humana, Clifton, N.J., 1991) vol. 7. pp.109-127). Expression of the inserted hedgehog gene can be under controlof, for example, the E1A promoter, the major late promoter (MLP) andassociated leader sequences, the E3 promoter, or exogenously addedpromoter sequences.

[0131] Yet another viral vector system useful for delivery of one of thesubject vertebrate hh genes is the adeno-associated virus (AAV).Adeno-associated virus is a naturally occurring defective virus thatrequires another virus, such as an adenovirus or a herpes virus, as ahelper virus for efficient replication and a productive life cycle (Fora review see Muzyczka et al. Curr. Topics in Micro. and Immunol. (1992)158:97-129). It is also one of the few viruses that may integrate itsDNA into non-dividing cells, and exhibits a high frequency of stableintegration (see for example Flotte et al. (1992) Am. J. Respir. Cell.Mol. Biol. 7:349-356; Samulski et al. (1989) J. Virol. 63:3822-3828; andMcLaughlin et al. (1989) J. Virol. 62:1963-1973). Vectors containing aslittle as 300 base pairs of AAV can be packaged and can integrate. Spacefor exogenous DNA is limited to about 4.5 kb. An AAV vector such as thatdescribed in Tratschin et al. (1985) Mol. Cell. Biol. 5:3251-3260 can beused to introduce DNA into cells. A variety of nucleic acids have beenintroduced into different cell types using AAV vectors (see for exampleHermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470;Tratschin et al. (1985) Mol. Cell. Biol. 4:2072-2081; Wondisford et al.(1988) Mol. Endocrinol. 2:32-39; Tratschin et al. (1984) J. Virol.51:611-619; and Flotte et al. (1993) J. Biol. Chem. 268:3781-3790).

[0132] In addition to viral transfer methods, such as those illustratedabove, non-viral methods can also be employed to cause expression of asubject hedgehog polypeptide in the tissue of an animal. Most nonviralmethods of gene transfer rely on normal mechanisms used by mammaliancells for the uptake and intracellular transport of macromolecules. Inpreferred embodiments, non-viral gene delivery systems of the presentinvention rely on endocytic pathways for the uptake of the subject hhpolypeptide gene by the targeted cell. Exemplary gene delivery systemsof this type include liposomal derived systems, poly-lysine conjugates,and artificial viral envelopes.

[0133] In clinical settings, the gene delivery systems for thetherapeutic hedgehog gene can be introduced into a patient by any of anumber of methods, each of which is familiar in the art. For instance, apharmaceutical preparation of the gene delivery system can be introducedsystemically, e.g. by intravenous injection, and specific transductionof the protein in the target cells occurs predominantly from specificityof transfection provided by the gene delivery vehicle, cell-type ortissue-type expression due to the transcriptional regulatory sequencescontrolling expression of the receptor gene, or a combination thereof.In other embodiments, initial delivery of the recombinant gene is morelimited with introduction into the animal being quite localized. Forexample, the gene delivery vehicle can be introduced by catheter (seeU.S. Pat. No. 5,328,470) or by stereotactic injection (e.g. Chen et al.(1994) PNAS 91: 3054-3057). A vertebrate hh gene, such as any one of theclones represented in the group consisting of SEQ ID NO:1-7, can bedelivered in a gene therapy construct by electroporation usingtechniques described, for example, by Dev et al. ((1994) Cancer TreatRev 20:105-115).

[0134] The pharmaceutical preparation of the gene therapy construct canconsist essentially of the gene delivery system in an acceptablediluent, or can comprise a slow release matrix in which the genedelivery vehicle is imbedded. Alternatively, where the complete genedelivery system can be produced intact from recombinant cells, e.g.retroviral vectors, the pharmaceutical preparation can comprise one ormore cells which produce the gene delivery system.

[0135] Another aspect of the present invention concerns recombinantforms of the hedgehog proteins. Recombinant polypeptides preferred bythe present invention, in addition to native hedgehog proteins, are atleast 60% homologous, more preferably 70% homologous and most preferably80% homologous with an amino acid sequence represented by any of SEQ IDNos:8-14. Polypeptides which possess an activity of a hedgehog protein(i.e. either agonistic or antagonistic), and which are at least 90%,more preferably at least 95%, and most preferably at least about 98-99%homologous with a sequence selected from the group consisting of SEQ IDNos:8-14 are also within the scope of the invention.

[0136] The term “recombinant protein” refers to a polypeptide of thepresent invention which is produced by recombinant DNA techniques,wherein generally, DNA encoding a vertebrate hh polypeptide is insertedinto a suitable expression vector which is in turn used to transform ahost cell to produce the heterologous protein. Moreover, the phrase“derived from”, with respect to a recombinant hedgehog gene, is meant toinclude within the meaning of “recombinant protein” those proteinshaving an amino acid sequence of a native hedgehog protein, or an aminoacid sequence similar thereto which is generated by mutations includingsubstitutions and deletions (including truncation) of a naturallyoccurring form of the protein.

[0137] The present invention further pertains to recombinant forms ofone of the subject hedgehog polypeptides which are encoded by genesderived from a vertebrate organism, particularly a mammal (e.g. ahuman), and which have amino acid sequences evolutionarily related tothe hedgehog proteins represented in SEQ ID Nos:8-14. Such recombinanthh polypeptides preferably are capable of functioning in one of eitherrole of an agonist or antagonist of at least one biological activity ofa wild-type (“authentic”) hedgehog protein of the appended sequencelisting. The term “evolutionarily related to”, with respect to aminoacid sequences of vertebrate hedgehog proteins, refers to bothpolypeptides having amino acid sequences which have arisen naturally,and also to mutational variants of vertebrate hh polypeptides which arederived, for example, by combinatorial mutagenesis. Such evolutionarilyderived hedgehog proteins polypeptides preferred by the presentinvention are at least 60% homologous, more preferably 70% homologousand most preferably 80% homologous with the amino acid sequence selectedfrom the group consisting of SEQ ID Nos:8-14. Polypeptides having atleast about 90%, more preferably at least about 95%, and most preferablyat least about 98-99% homology with a sequence selected from the groupconsisting of SEQ ID Nos:8-14 are also within the scope of theinvention.

[0138] The present invention further pertains to methods of producingthe subject hedgehog polypeptides. For example, a host cell transfectedwith a nucleic acid vector directing expression of a nucleotide sequenceencoding the subject polypeptides can be cultured under appropriateconditions to allow expression of the peptide to occur. The polypeptidehedgehog may be secreted and isolated from a mixture of cells and mediumcontaining the recombinant vertebrate hh polypeptide. Alternatively, thepeptide may be retained cytoplasmically by removing the signal peptidesequence from the recombinant hh gene and the cells harvested, lysed andthe protein isolated. A cell culture includes host cells, media andother byproducts. Suitable media for cell culture are well known in theart. The recombinant hh polypeptide can be isolated from cell culturemedium, host cells, or both using techniques known in the art forpurifying proteins including ion-exchange chromatography, gel filtrationchromatography, ultrafiltration, electrophoresis, and immunoaffinitypurification with antibodies specific for such peptide. In a preferredembodiment, the recombinant hh polypeptide is a fusion proteincontaining a domain which facilitates its purification, such as anhh/GST fusion protein.

[0139] This invention also pertains to a host cell transfected toexpress a recombinant form of the subject hedgehog polypeptides. Thehost cell may be any prokaryotic or eukaryotic cell. Thus, a nucleotidesequence derived from the cloning of vertebrate hedgehog proteins,encoding all or a selected portion of the full-length protein, can beused to produce a recombinant form of a vertebrate hh polypeptide viamicrobial or eukaryotic cellular processes. Ligating the polynucleotidesequence into a gene construct, such as an expression vector, andtransforming or transfecting into hosts, either eukaryotic (yeast,avian, insect or mammalian) or prokaryotic (bacterial cells), arestandard procedures used in producing other well-known proteins, e.g.insulin, interferons, human growth hormone, IL-1, IL-2, and the like.Similar procedures, or modifications thereof, can be employed to preparerecombinant hedgehog polypeptides by microbial means or tissue-culturetechnology in accord with the subject invention.

[0140] The recombinant hedgehog genes can be produced by ligatingnucleic acid encoding an hh protein, or a portion thereof, into a vectorsuitable for expression in either prokaryotic cells, eukaryotic cells,or both. Expression vectors for production of recombinant forms of thesubject hh polypeptides include plasmids and other vectors. Forinstance, suitable vectors for the expression of a hedgehog polypeptideinclude plasmids of the types: pBR322-derived plasmids, pEMBL-derivedplasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derivedplasmids for expression in prokaryotic cells, such as E. coli.

[0141] A number of vectors exist for the expression of recombinantproteins in yeast. For instance, YEP24, YIP5, YEP51, YEP52, pYES2, andYRP17 are cloning and expression vehicles useful in the introduction ofgenetic constructs into S. cerevisiae (see, for example, Broach et al.(1983) in Experimental Manipulation of Gene Expression, ed. M. InouyeAcademic Press, p. 83, incorporated by reference herein). These vectorscan replicate in E. coli due the presence of the pBR322 ori, and in S.cerevisiae due to the replication determinant of the yeast 2 micronplasmid. In addition, drug resistance markers such as ampicillin can beused. In an illustrative embodiment, an hh polypeptide is producedrecombinantly utilizing an expression vector generated by sub-cloningthe coding sequence of one of the hedgehog genes represented in SEQ IDNos:1-7.

[0142] The preferred mammalian expression vectors contain bothprokaryotic sequences, to facilitate the propagation of the vector inbacteria, and one or more eukaryotic transcription units that areexpressed in eukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV,pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo andpHyg derived vectors are examples of mammalian expression vectorssuitable for transfection of eukaryotic cells. Some of these vectors aremodified with sequences from bacterial plasmids, such as pBR322, tofacilitate replication and drug resistance selection in both prokaryoticand eukaryotic cells. Alternatively, derivatives of viruses such as thebovine papillomavirus (BPV-1), or Epstein-Barr virus (pHEBo,pREP-derived and p205) can be used for transient expression of proteinsin eukaryotic cells. The various methods employed in the preparation ofthe plasmids and transformation of host organisms are well known in theart. For other suitable expression systems for both prokaryotic andeukaryotic cells, as well as general recombinant procedures, seeMolecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritschand Maniatis (Cold Spring Harbor Laboratory Press: 1989) Chapters 16 and17.

[0143] In some instances, it may be desirable to express the recombinanthedgehog polypeptide by the use of a baculovirus expression system.Examples of such baculovirus expression systems include pVL-derivedvectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derived vectors(such as pAcUW1), and pBlueBac-derived vectors (such as the β-galcontaining pBlueBac III).

[0144] When it is desirable to express only a portion of an hh protein,such as a form lacking a portion of the N-terminus, i.e. a truncationmutant which lacks the signal peptide, it may be necessary to add astart codon (ATG) to the oligonucleotide fragment containing the desiredsequence to be expressed. It is well known in the art that a methionineat the N-terminal position can be enzymatically cleaved by the use ofthe enzyme methionine aminopeptidase (MAP). MAP has been cloned from E.coli (Ben-Bassat et al. (1987) J. Bacteriol. 169:751-757) and Salmonellatyphimurium and its in vitro activity has been demonstrated onrecombinant proteins (Miller et al. (1987) PNAS 84:2718-1722).Therefore, removal of an N-terminal methionine, if desired, can beachieved either in vivo by expressing hedgehog-derived polypeptides in ahost which produces MAP (e.g., E. coli or CM89 or S. cerevisiae), or invitro by use of purified MAP (e.g., procedure of Miller et al., supra).

[0145] Alternatively, the coding sequences for the polypeptide can beincorporated as a part of a fusion gene including a nucleotide sequenceencoding a different polypeptide. This type of expression system can beuseful under conditions where it is desirable to produce an immunogenicfragment of a hedgehog protein. For example, the VP6 capsid protein ofrotavirus can be used as an immunologic carrier protein for portions ofthe hh polypeptide, either in the monomeric form or in the form of aviral particle. The nucleic acid sequences corresponding to the portionof a subject hedgehog protein to which antibodies are to be raised canbe incorporated into a fusion gene construct which includes codingsequences for a late vaccinia virus structural protein to produce a setof recombinant viruses expressing fusion proteins comprising hh epitopesas part of the virion. It has been demonstrated with the use ofimmunogenic fusion proteins utilizing the Hepatitis B surface antigenfusion proteins that recombinant Hepatitis B virions can be utilized inthis role as well. Similarly, chimeric constructs coding for fusionproteins containing a portion of an hh protein and the poliovirus capsidprotein can be created to enhance immunogenicity of the set ofpolypeptide antigens (see, for example, EP Publication No: 0259149; andEvans et al. (1989) Nature 339:385; Huang et al. (1988) J. Virol.62:3855; and Schlienger et al. (1992) J. Virol. 66:2).

[0146] The Multiple Antigen Peptide system for peptide-basedimmunization can also be utilized to generate an immunogen, wherein adesired portion of an hh polypeptide is obtained directly fromorgano-chemical synthesis of the peptide onto an oligomeric branchinglysine core (see, for example, Posnett et al. (1988) JBC 263:1719 andNardelli et al. (1992) J. Immunol. 148:914). Antigenic determinants ofhh proteins can also be expressed and presented by bacterial cells.

[0147] In addition to utilizing fusion proteins to enhanceimmunogenicity, it is widely appreciated that fusion proteins can alsofacilitate the expression of proteins, and accordingly, can be used inthe expression of the vertebrate hh polypeptides of the presentinvention. For example, hedgehog polypeptides can be generated asglutathione-S-transferase (GST-fusion) proteins. Such GST-fusionproteins can enable easy purification of the hedgehog polypeptide, asfor example by the use of glutathione-derivatized matrices (see, forexample, Current Protocols in Molecular Biology, eds. Ausubel et al.(N.Y.: John Wiley & Sons, 1991)). In another embodiment, a fusion genecoding for a purification leader sequence, such as apoly-(His)/enterokinase cleavage site sequence, can be used to replacethe signal sequence which naturally occurs at the N-terminus of the hhprotein (e.g.of the pro-form, in order to permit purification of thepoly(His)-hh protein by affinity chromatography using a Ni²⁺ metalresin. The purification leader sequence can then be subsequently removedby treatment with enterokinase (e.g., see Hochuli et al. (1987) J.Chromatography 411:177; and Janknecht et al. PNAS 88:8972).

[0148] Techniques for making fusion genes are known to those skilled inthe art. Essentially, the joining of various DNA fragments coding fordifferent polypeptide sequences is performed in accordance withconventional techniques, employing blunt-ended or stagger-ended terminifor ligation, restriction enzyme digestion to provide for appropriatetermini, filling-in of cohesive ends as appropriate, alkalinephosphatase treatment to avoid undesirable joining, and enzymaticligation. In another embodiment, the fusion gene can be synthesized byconventional techniques including automated DNA synthesizers.Alternatively, PCR amplification of gene fragments can be carried outusing anchor primers which give rise to complementary overhangs betweentwo consecutive gene fragments which can subsequently be annealed togenerate a chimeric gene sequence (see, for example, Current Protocolsin Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).

[0149] Hedgehog polypeptides may also be chemically modified to createhh derivatives by forming covalent or aggregate conjugates with otherchemical moieties, such as glycosyl groups, lipids, phosphate, acetylgroups and the like. Covalent derivatives of hedgehog proteins can beprepared by linking the chemical moieties to functional groups on aminoacid sidechains of the protein or at the N-terminus or at the C-terminusof the polypeptide.

[0150] For instance, hedgehog proteins can be generated to include amoiety, other than sequence naturally associated with the protein, thatbinds a component of the extracellular matrix and enhances localizationof the analog to cell surfaces. For example, sequences derived from thefibronectin “type-III repeat”, such as a tetrapeptide sequence R-G-D-S(Pierschbacher et al. (1984) Nature 309:30-3; and Kornblihtt et al.(1985) EMBO 4:1755-9) can be added to the hh polypeptide to supportattachment of the chimeric molecule to a cell through binding ECMcomponents (Ruoslahti et al. (1987) Science 238:491-497; Pierschbacheretal. (1987) J. Biol. Chem. 262:17294-8.; Hynes (1987) Cell 48:549-54; andHynes (1992) Cell 69:11-25).

[0151] The present invention also makes available isolated hedgehogpolypeptides which are isolated from, or otherwise substantially free ofother cellular and extracellular proteins, especially morphogenicproteins or other extracellular or cell surface associated proteinswhich may normally be associated with the hedgehog polypeptide. The term“substantially free of other cellular or extracellular proteins” (alsoreferred to herein as “contaminating proteins”) or “substantially pureor purified preparations” are defined as encompassing preparations of hhpolypeptides having less than 20% (by dry weight) contaminating protein,and preferably having less than 5% contaminating protein. Functionalforms of the subject polypeptides can be prepared, for the first time,as purified preparations by using a cloned gene as described herein. By“purified”, it is meant, when referring to a peptide or DNA or RNAsequence, that the indicated molecule is present in the substantialabsence of other biological macromolecules, such as other proteins. Theterm “purified” as used herein preferably means at least 80% by dryweight, more preferably in the range of 95-99% by weight, and mostpreferably at least 99.8% by weight, of biological macromolecules of thesame type present (but water, buffers, and other small molecules,especially molecules having a molecular weight of less than 5000, can bepresent). The term “pure” as used herein preferably has the samenumerical limits as “purified” immediately above. “Isolated” and“purified” do not encompass either natural materials in their nativestate or natural materials that have been separated into components(e.g., in an acrylamide gel) but not obtained either as pure (e.g.lacking contaminating proteins, or chromatography reagents such asdenaturing agents and polymers, e.g. acrylamide or agarose) substancesor solutions. In preferred embodiments, purified hedgehog preparationswill lack any contaminating proteins from the same animal from thathedgehog is normally produced, as can be accomplished by recombinantexpression of, for example, a human hedgehog protein in a non-humancell.

[0152] As described above for recombinant polypeptides, isolated hhpolypeptides can include all or a portion of the amino acid sequencesrepresented in SEQ ID No:8, SEQ ID No:9, SEQ ID No:10, SEQ ID No:11, SEQID No:12, SEQ ID No:13 or SEQ ID No:14, or a homologous sequencethereto. Preferred fragments of the subject hedgehog proteins correspondto the N-terminal and C-terminal proteolytic fragments of the matureprotein (see, for instance, Examples 6 and 9). Bioactive fragments ofhedgehog polypeptides are described in great detail in U.S. Ser. No.08/435,093, filed May 4, 1995, herein incorporated by reference.

[0153] Isolated peptidyl portions of hedgehog proteins can be obtainedby screening peptides recombinantly produced from the correspondingfragment of the nucleic acid encoding such peptides. In addition,fragments can be chemically synthesized using techniques known in theart such as conventional Merrifield solid phase f-Moc or t-Bocchemistry. For example, a hedgehog polypeptide of the present inventionmay be arbitrarily divided into fragments of desired length with nooverlap of the fragments, or preferably divided into overlappingfragments of a desired length. The fragments can be produced(recombinantly or by chemical synthesis) and tested to identify thosepeptidyl fragments which can function as either agonists or antagonistsof a wild-type (e.g., “authentic”) hedgehog protein.

[0154] The recombinant hedgehog polypeptides of the present inventionalso include homologs of the authentic hedgehog proteins, such asversions of those protein which are resistant to proteolytic cleavage,as for example, due to mutations which alter potential cleavagesequences or which inactivate an enzymatic activity associated with theprotein. Hedgehog homologs of the present invention also includeproteins which have been post-translationally modified in a mannerdifferent than the authentic protein. Exemplary derivatives ofvertebrate hedgehog proteins include polypeptides which lackN-glycosylation sites (e.g. to produce an unglycosylated protein), orwhich lack N-terminal and/or C-terminal sequences.

[0155] Modification of the structure of the subject vertebrate hhpolypeptides can be for such purposes as enhancing therapeutic orprophylactic efficacy, or stability (e.g., ex vivo shelf life andresistance to proteolytic degradation in vivo). Such modified peptides,when designed to retain at least one activity of the naturally-occurringform of the protein, are considered functional equivalents of thehedgehog polypeptides described in more detail herein. Such modifiedpeptides can be produced, for instance, by amino acid substitution,deletion, or addition.

[0156] For example, it is reasonable to expect that an isolatedreplacement of a leucine with an isoleucine or valine, an aspartate witha glutamate, a threonine with a serine, or a similar replacement of anamino acid with a structurally related amino acid (i.e. isosteric and/orisoelectric mutations) will not have a major effect on the biologicalactivity of the resulting molecule. Conservative replacements are thosethat take place within a family of amino acids that are related in theirside chains. Genetically encoded amino acids are can be divided intofour families: (1) acidic=aspartate, glutamate; (2) basic=lysine,arginine, histidine; (3) nonpolar=alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan; and (4) unchargedpolar=glycine, asparagine, glutamine, cysteine, serine, threonine,tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimesclassified jointly as aromatic amino acids. In similar fashion, theamino acid repertoire can be grouped as (1) acidic=aspartate, glutamate;(2) basic=lysine, arginine histidine, (3) aliphatic=glycine, alanine,valine, leucine, isoleucine, serine, threonine, with serine andthreonine optionally be grouped separately as aliphatic-hydroxyl; (4)aromatic=phenylalanine, tyrosine, tryptophan; (5) amide=asparagine,glutamine; and (6) sulfur-containing=cysteine and methionine. (see, forexample, Biochemistry, 2nd ed., Ed. by L. Stryer, W H Freeman and Co.:1981). Whether a change in the amino acid sequence of a peptide resultsin a functional hedgehog homolog (e.g. functional in the sense that itacts to mimic or antagonize the wild-type form) can be readilydetermined by assessing the ability of the variant peptide to produce aresponse in cells in a fashion similar to the wild-type protein, orcompetitively inhibit such a response. Polypeptides in which more thanone replacement has taken place can readily be tested in the samemanner.

[0157] This invention further contemplates a method for generating setsof combinatorial mutants of the subject hedgehog proteins as well astruncation mutants, and is especially useful for identifying potentialvariant sequences (e.g. homologs) that are functional in binding to areceptor for hedgehog proteins. The purpose of screening suchcombinatorial libraries is to generate, for example, novel hh homologswhich can act as either agonists or antagonist, or alternatively,possess novel activities all together. To illustrate, hedgehog homologscan be engineered by the present method to provide more efficientbinding to a cognate receptor, yet still retain at least a portion of anactivity associated with hh. Thus, combinatorially-derived homologs canbe generated to have an increased potency relative to a naturallyoccurring form of the protein. Likewise, hedgehog homologs can begenerated by the present combinatorial approach to act as antagonists,in that they are able to mimic, for example, binding to otherextracellular matrix components (such as receptors), yet not induce anybiological response, thereby inhibiting the action of authentic hedgehogor hedgehog agonists. Moreover, manipulation of certain domains of hh bythe present method can provide domains more suitable for use in fusionproteins, such as one that incorporates portions of other proteins whichare derived from the extracellular matrix and/or which bindextracellular matrix components.

[0158] In one aspect of this method, the amino acid sequences for apopulation of hedgehog homologs or other related proteins are aligned,preferably to promote the highest homology possible. Such a populationof variants can include, for example, hh homologs from one or morespecies. Amino acids which appear at each position of the alignedsequences are selected to create a degenerate set of combinatorialsequences. In a preferred embodiment, the variegated library of hedgehogvariants is generated by combinatorial mutagenesis at the nucleic acidlevel, and is encoded by a variegated gene library. For instance, amixture of synthetic oligonucleotides can be enzymatically ligated intogene sequences such that the degenerate set of potential hh sequencesare expressible as individual polypeptides, or alternatively, as a setof larger fusion proteins (e.g. for phage display) containing the set ofhh sequences therein.

[0159] As illustrated in FIG. 5A, to analyze the sequences of apopulation of variants, the amino acid sequences of interest can bealigned relative to sequence homology. The presence or absence of aminoacids from an aligned sequence of a particular variant is relative to achosen consensus length of a reference sequence, which can be real orartificial. In order to maintain the highest homology in alignment ofsequences, deletions in the sequence of a variant relative to thereference sequence can be represented by an amino acid space ( or *),while insertional mutations in the variant relative to the referencesequence can be disregarded and left out of the sequence of the variantwhen aligned. For instance, FIG. 5A includes the alignment of severalcloned forms of hh from different species. Analysis of the alignment ofthe hh clones shown in FIG. 5A can give rise to the generation of adegenerate library of polypeptides comprising potential hh sequences.

[0160] In an illustrative embodiment, alignment of exons 1, 2 and aportion of exon 3 encoded sequences (e.g. the N-terminal approximately221 residues of the mature protein) of each of the Shh clones produces adegenerate set of Shh polypeptides represented by the general formula:(SEQ ID No: 40) C-G-P-G-R-G-X(1) -G -X(2)-R-R-H-P-K-K-L-T-P-L-A-Y-K-Q-F-I-P-N-V-A-E-K-T-L-G-A-S-G-R-Y-E-G-K-I-X(3) -R-N-S-E-R-F-K-E-L-T-P-N-Y-N-P-D-I-I-F-K-D-E-E-N-T-G-A-D-R-L-M-T-Q-R-C-K-D-K-L-N-X(4) -L-A-I-S-V-M-N-X(5) -W-P-G-V-X(6) -L-R-V-T-E-G-W-D-E-D-G-H-H-X(7) -E-E-S-L-H-Y-E-G-R-A-V-D-I-T-T-S-D-R-D-X(8) -S-K-Y-G -X(9) -L-X(10) -R-LA-V-E-A-G-F-D-W-V-Y-Y-E-S-K-A-H-I-H-C-S-V-K-A-E-N-S-V-A-A-K-S-G-G-C-F-P-G-S-A-X(11) -V-X(12) -L-X(13) -X(14) -G-G-X(15) -K-X- (16)-V-K-D-L-X(17) -P-G-D-X(18) -V-L-A-A-D-X(19) -X(20) -G-X(21) -L- X(22)-X(23) -S-D-F-X(24) -X(25) -F-X(26) -D-R ,

[0161] wherein each of the degenerate positions “X” can be an amino acidwhich occurs in that position in one of the human, mouse, chicken orzebrafish Shh clones, or, to expand the library, each X can also beselected from amongst amino acid residue which would be conservativesubstitutions for the amino acids which appear naturally in each ofthose positions. For instance, Xaa(1) represents Gly, Ala, Val, Leu,Ile, Phe, Tyr or Trp; Xaa(2) represents Arg, His or Lys; Xaa(3)represents Gly, Ala, Val, Leu, Ile, Ser or Thr; Xaa(4) represents Gly,Ala, Val, Leu, Ile, Ser or Thr; Xaa(5) represents Lys, Arg, His, Asn orGln; Xaa(6) represents Lys, Arg or His; Xaa(7) represents Ser, Thr, Tyr,Trp or Phe; Xaa(8) represents Lys, Arg or His; Xaa(9) represents Met,Cys, Ser or Thr; Xaa(10) represents Gly, Ala, Val, Leu, Ile, Ser or Thr;Xaa(11) represents Leu, Val, Met, Thr or Ser; Xaa(12) represents His,Phe, Tyr, Ser, Thr, Met or Cys; Xaa(13) represents Gln, Asn, Glu, orAsp; Xaa(14) represents His, Phe, Tyr, Thr, Gln, Asn, Glu or Asp;Xaa(15) represents Gln, Asn, Glu, Asp, Thr, Ser, Met or Cys; Xaa(16)represents Ala, Gly, Cys, Leu, Val or Met; Xaa(17) represents Arg, Lys,Met, Ile, Asn, Asp, Glu, Gln, Ser, Thr or Cys; Xaa(18) represents Arg,Lys, Met or Ile; Xaa(19) represents Ala, Gly, Cys, Asp, Glu, Gln, Asn,Ser, Thr or Met; Xaa(20) represents Ala, Gly, Cys, Asp, Asn, Glu or Gln;Xaa(21) represents Arg, Lys, Met, Ile, Asn, Asp, Glu or Gln; Xaa(22)represent Leu, Val, Met or Ile; Xaa(23) represents Phe, Tyr, Thr, His orTrp; Xaa(24) represents Ile, Val, Leu or Met; .Xaa(25) represents Met,Cys, Ile, Leu, Val, Thr or Ser; Xaa(26) represents Leu, Val, Met, Thr orSer. In an even more expansive library, each X can be selected from anyamino acid.

[0162] In similar fashion, alignment of each of the human, mouse,chicken and zebrafish hedgehog clones (FIG. 5B), can provide adegenerate polypeptide sequence represented by the general formula: (SEQID No: 41) C-G-P-G-R-G-X(1) -X(2) -X(3) -R-R-X(4) -X(5) -X(6) -P-K-X(7)-L-X(8) - P-L-X(9) -Y-K-Q-F-X(10) -P-X(11) -X(12) -X(13) -E-X(14)-T-L-G-A-S-G- X(15) -X(16) -E-G-X(17) -X(18) -X(19) -R-X(20)-S-E-R-F-X(21) -X(22) - L-T-P-N-Y-N-P-D-I-I-F-K-D-E-E-N -X(23)-G-A-D-R-L-M-T-X(24) -R-C- K-X(25) -X(26) -X(27) -N-X(28)-L-A-I-S-V-M-N-X(29) -W-P-G-V-X(30) - L-R-V-T-E-G-X(31)-D-E-D-G-H-H-X(32) -X(33) -X(34) -S-L-H-Y-E-G-R- A-X(35)-D-I-T-T-S-D-R-D-X(36) -X(37) -K-Y-G-X(38) -L-X(39) -R-L-A-V-E-A-G-F-D-W-V-Y-Y-E-S-X(40) -X(41) -H-X(42) -H-X(43) -S-V-K-X(44)-X(45),

[0163] wherein, as above, each of the degenerate positions “X” can be anamino acid which occurs in a corresponding position in one of thewild-type clones, and may also include amino acid residue which would beconservative substitutions, or each X can be any amino acid residue. Inan exemplary embodiment, Xaa(1) represents Gly, Ala, Val, Leu, Ile, Pro,Phe or Tyr; Xaa(2) represents Gly, Ala, Val, Leu or Ile; Xaa(3)represents Gly, Ala, Val, Leu, Ile, Lys, His or Arg; Xaa(4) representsLys, Arg or His; Xaa(5) represents Phe, Trp, Tyr or an amino acid gap;Xaa(6) represents Gly, Ala, Val, Leu, Ile or an amino acid gap; Xaa(7)represents Asn, Gln, His, Arg or Lys; Xaa(8) represents Gly, Ala, Val,Leu, Ile, Ser or Thr; Xaa(9) represents Gly, Ala, Val, Leu, Ile, Ser orThr; Xaa(10) represents Gly, Ala, Val, Leu, Ile, Ser or Thr; Xaa(11)represents Ser, Thr, Gln or Asn; Xaa(12) represents Met, Cys, Gly, Ala,Val, Leu, Ile, Ser or Thr; Xaa(13) represents Gly, Ala, Val, Leu, Ile orPro; Xaa(14) represents Arg, His or Lys; Xaa(15) represents Gly, Ala,Val, Leu, Ile, Pro, Arg, His or Lys; Xaa(16) represents Gly, Ala, Val,Leu, Ile, Phe or Tyr; Xaa(17) represents Arg, His or Lys; Xaa(18)represents Gly, Ala, Val, Leu, Ile, Ser or Thr; Xaa(19) represents Thror Ser; Xaa(20) represents Gly, Ala, Val, Leu, Ile, Asn or Gln; Xaa(21)represents Arg, His or Lys; Xaa(22) represents Asp or Glu; Xaa(23)represents Ser or Thr; Xaa(24) represents Glu, Asp, Gln or Asn; Xaa(25)represents Glu or Asp; Xaa(26) represents Arg, His or Lys; Xaa(27)represents Gly, Ala, Val, Leu or Ile; Xaa(28) represents Gly, Ala, Val,Leu, Ile, Thr or Ser; Xaa(29) represents Met, Cys, Gln, Asn, Arg, Lys orHis; Xaa(30) represents Arg, His or Lys; Xaa(31) represents Trp, Phe,Tyr, Arg, His or Lys; Xaa(32) represents Gly, Ala, Val, Leu, Ile, Ser,Thr, Tyr or Phe; Xaa(33) represents Gln, Asn, Asp or Glu; Xaa(34)represents Asp or Glu; Xaa(35) represents Gly, Ala, Val, Leu, or Ile;Xaa(36) represents Arg, His or Lys; Xaa(37) represents Asn, Gln, Thr orSer; Xaa(38) represents Gly, Ala, Val, Leu, Ile, Ser, Thr, Met or Cys;Xaa(39) represents Gly, Ala, Val, Leu, Ile, Thr or Ser; Xaa(40)represents Arg, His or Lys; Xaa(41) represents Asn, Gln, Gly, Ala, Val,Leu or Ile; Xaa(42) represents Gly, Ala, Val, Leu or Ile; Xaa(43)represents Gly, Ala, Val, Leu, Ile, Ser, Thr or Cys; Xaa(44) representsGly, Ala, Val, Leu, Ile, Thr or Ser; and Xaa(45) represents Asp or Glu.

[0164] There are many ways by which the library of potential hh homologscan be generated from a degenerate oligonucleotide sequence. Chemicalsynthesis of a degenerate gene sequence can be carried out in anautomatic DNA synthesizer, and the synthetic genes then ligated into anappropriate expression vector. The purpose of a degenerate set of genesis to provide, in one mixture, all of the sequences encoding the desiredset of potential hh sequences. The synthesis of degenerateoligonucleotides is well known in the art (see for example, Narang, S A(1983) Tetrahedron 39:3; Itakura et al. (1981) Recombinant DNA, Proc 3rdCleveland Sympos. Macromolecules, ed. A G Walton, Amsterdam: Elsevierpp273-289; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura etal. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477.Such techniques have been employed in the directed evolution of otherproteins (see, for example, Scott et al. (1990) Science 249:386-390;Roberts et al. (1992) PNAS 89:2429-2433; Devlin et al. (1990) Science249: 404-406; Cwirla et al. (1990) PNAS 87: 6378-6382; as well as U.S.Pat. Nos. 5,223,409, 5,198,346, and 5,096,815).

[0165] A wide range of techniques are known in the art for screeninggene products of combinatorial libraries made by point mutations, andfor screening cDNA libraries for gene products having a certainproperty. Such techniques will be generally adaptable for rapidscreening of the gehe libraries generated by the combinatorialmutagenesis of hedgehog homologs. The most widely used techniques forscreening large gene libraries typically comprises cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions in which detection of a desiredactivity facilitates relatively easy isolation of the vector encodingthe gene whose product was detected. Each of the illustrative assaysdescribed below are amenable to high through-put analysis as necessaryto screen large numbers of degenerate hedgehog sequences created bycombinatorial mutagenesis techniques.

[0166] In one embodiment, the combinatorial library is designed to besecreted (e.g. the polypeptides of the library all include a signalsequence but no transmembrane or cytoplasmic domains), and is used totransfect a eukaryotic cell that can be co-cultured with embryoniccells. A functional hedgehog protein secreted by the cells expressingthe combinatorial library will diffuse to neighboring embryonic cellsand induce a particular biological response, such as to illustrate,neuronal differentiation. Using antibodies directed to epitopes ofparticular neuronal cells (e.g. Islet-1 or Pax-1), the pattern ofdetection of neuronal induction will resemble a gradient function, andwill allow the isolation (generally after several repetitive rounds ofselection) of cells producing active hedgehog homologs. Likewise, hhantagonists can be selected in similar fashion by the ability of thecell producing a functional antagonist to protect neighboring cells fromthe effect of wild-type hedgehog added to the culture media.

[0167] To illustrate, target cells are cultured in 24-well microtitreplates. Other eukaryotic cells are transfected with the combinatorial hhgene library and cultured in cell culture inserts (e.g. CollaborativeBiomedical Products, Catalog #40446) that are able to fit into the wellsof the microtitre plate. The cell culture inserts are placed in thewells such that recombinant hh homologs secreted by the cells in theinsert can diffuse through the porous bottom of the insert and contactthe target cells in the microtitre plate wells. After a period of timesufficient for functional forms of a hedgehog protein to produce ameasurable response in the target cells, the inserts are removed and theeffect of the variant hedgehog proteins on the target cells determined.For example, where the target cell is a neural crest cell and theactivity desired from the hh homolog is the induction of neuronaldifferentiation, then fluorescently-labeled antibodies specific forIslet-1 or other neuronal markers can be used to score for induction inthe target cells as indicative of a functional hh in that well. Cellsfrom the inserts corresponding to wells which score positive foractivity can be split and re-cultured on several inserts, the processbeing repeated until the active clones are identified.

[0168] In yet another screening assay, the candidate hedgehog geneproducts are displayed on the surface of a cell or viral particle, andthe ability of particular cells or viral particles to associate with ahedgehog-binding moiety (such as an hedgehog receptor or a ligand whichbinds the hedgehog protein) via this gene product is detected in a“panning assay”. Such panning steps can be carried out on cells culturedfrom embryos. For instance, the gene library can be cloned into the genefor a surface membrane protein of a bacterial cell, and the resultingfusion protein detected by panning (Ladner et al., WO 88/06630; Fuchs etal. (1991) Bio/Technology 9:1370-1371; and Goward et al. (1992) TIBS18:136-140). In a similar fashion, fluorescently labeled molecules whichbind hh can be used to score for potentially functional hh homologs.Cells can be visually inspected and separated under a fluorescencemicroscope, or, where the morphology of the cell permits, separated by afluorescence-activated cell sorter.

[0169] In an alternate embodiment, the gene library is expressed as afusion protein on the surface of a viral particle. For instance, in thefilamentous phage system, foreign peptide sequences can be expressed onthe surface of infectious phage, thereby conferring two significantbenefits. First, since these phage can be applied to affinity matricesat very high concentrations, large number of phage can be screened atone time. Second, since each infectious phage displays the combinatorialgene product on its surface, if a particular phage is recovered from anaffinity matrix in low yield, the phage can be amplified by anotherround of infection. The group of almost identical E. coli filamentousphages M13, fd, and f1 are most often used in phage display libraries,as either of the phage gIII or gVIII coat proteins can be used togenerate fusion proteins without disrupting the ultimate packaging ofthe viral particle (Ladner et al. PCT publication WO 90/02909; Garrardet al., PCT publication WO 92/09690; Marks et al. (1992) J. Biol. Chem.267:16007-16010; Griffths et al. (1993) EMBO J 12:725-734; Clackson etal. (1991) Nature 352:624-628; and Barbas et al. (1992) PNAS89:4457-4461).

[0170] In an illustrative embodiment, the recombinant phage antibodysystem (RPAS, Pharamacia Catalog number 27-9400-01) can be easilymodified for use in expressing and screening hh combinatorial libraries.For instance, the pCANTAB 5 phagemid of the RPAS kit contains the genewhich encodes the phage gIII coat protein. The hh combinatorial genelibrary can be cloned into the phagemid adjacent to the gIII signalsequence such that it will be expressed as a gIII fusion protein. Afterligation, the phagemid is used to transform competent E. coli TG1 cells.Transformed cells are subsequently infected with M13KO7 helper phage torescue the phagemid and its candidate hh gene insert. The resultingrecombinant phage contain phagemid DNA encoding a specific candidate hh,and display one or more copies of the corresponding fusion coat protein.The phage-displayed candidate hedgehog proteins which are capable ofbinding an hh receptor are selected or enriched by panning. Forinstance, the phage library can be applied to cultured embryonic cellsand unbound phage washed away from the cells. The bound phage is thenisolated, and if the recombinant phage express at least one copy of thewild type gIII coat protein, they will retain their ability to infect E.coli. Thus, successive rounds of reinfection of E. coli, and panningwill greatly enrich for hh homologs, which can then be screened forfurther biological activities in order to differentiate agonists andantagonists.

[0171] Combinatorial mutagenesis has a potential to generate very largelibraries of mutant proteins, e.g., in the order of 10²⁶ molecules.Combinatorial libraries of this size may be technically challenging toscreen even with high throughput screening assays such as phage display.To overcome this problem, a new technique has been developed recently,recrusive ensembel mutagenesis (REM), which allows one to avoid the veryhigh proportion of non-functional proteins in a random library andsimply enhances the frequency of functional proteins, thus decreasingthe complexity required to achieve a useful sampling of sequence space.REM is an algorithm which enhances the frequency of functional mutantsin a library when an appropriate selection or screening method isemployed (Arkin and Yourvan, 1992, PNAS USA 89:7811-7815; Yourvan etal., 1992, Parallel Problem Solvingfrom Nature, 2., In Maenner andManderick, eds., Elsevir Publishing Co., Amsterdam, pp. 401-410;Delgrave et al., 1993, Protein Engineering 6(3):327-331).

[0172] The invention also provides for reduction of the vertebrate hhprotein to generate mimetics, e.g. peptide or non-peptide agents, whichare able to disrupt binding of a vertebrate hh polypeptide of thepresent invention with an hh receptor. Thus, such mutagenic techniquesas described above are also useful to map the determinants of thehedgehog proteins which participate in protein-protein interactionsinvolved in, for example, binding of the subject vertebrate hhpolypeptide to other extracellular matrix components. To illustrate, thecritical residues of a subject hh polypeptide or hh ligand which areinvolved in molecular recognition of an hh receptor can be determinedand used to generate hedgehog-derived peptidomimetics whichcompetitively inhibit binding of the authentic hedgehog protein withthat moiety. By employing, for example, scanning mutagenesis to map theamino acid residues of each of the subject hedgehog proteins which areinvolved in binding other extracellular proteins, peptidomimeticcompounds can be generated which mimic those residues of the hedgehogprotein which facilitate the interaction. Such mimetics may then be usedto interfere with the normal function of a hedgehog protein Forinstance, non-hydrolyzable peptide analogs of such residues can begenerated using benzodiazepine (e.g., see Freidinger et al. in Peptides:Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden,Netherlands, 1988), azepine (e.g., see Huffman et al. in Peptides:Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden,Netherlands, 1988), substituted gama lactam rings (Garvey et al. inPeptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher:Leiden, Netherlands, 1988), keto-methylene pseudopeptides (Ewenson etal. (1986) J Med Chem 29:295; and Ewenson et al. in Peptides: Structureand Function (Proceedings of the 9th American Peptide Symposium) PierceChemical Co. Rockland, Ill., 1985), β-turn dipeptide cores (Nagai et al.(1985) Tetrahedron Lett 26:647; and Sato et al. (1986) J Chem Soc PerkinTrans 1:1231), and β-aminoalcohols (Gordon et al. (1985) Biochem BiophysRes Commun126:419; and Dann et al. (1986) Biochem Biophys Res Commun134:71).

[0173] Another aspect of the invention pertains to an antibodyspecifically reactive with a vertebrate hedgehog protein. For example,by using immunogens derived from hedgehog protein, e.g. based on thecDNA sequences, anti-protein/anti-peptide antisera or monoclonalantibodies can be made by standard protocols (See, for example,Antibodies: A Laboratory Manual ed. by Harlow and Lane (Cold SpringHarbor Press: 1988)). A mammal, such as a mouse, a hamster or rabbit canbe immunized with an immunogenic form of the peptide (e.g., a vertebratehh polypeptide or an antigenic fragment which is capable of eliciting anantibody response). Techniques for conferring immunogenicity on aprotein or peptide include conjugation to carriers or other techniqueswell known in the art. An immunogenic portion of a hedgehog protein canbe administered in the presence of adjuvant. The progress ofimmunization can be monitored by detection of antibody titers in plasmaor serum. Standard ELISA or other immunoassays can be used with theimmunogen as antigen to assess the levels of antibodies. In a preferredembodiment, the subject antibodies are immunospecific for antigenicdeterminants of a hedgehog protein of a vertebrate organism, such as amammal, e.g. antigenic determinants of a protein represented by SEQ IDNos:8-14 or a closely related homolog (e.g. at least 85% homologous,preferably at least 90% homologous, and more preferably at least 95%homologous). In yet a further preferred embodiment of the presentinvention, in order to provide, for example, antibodies which areimmuno-selective for discrete hedgehog homologs, e.g. Shh versus Dhhversus Ihh, the anti-hh polypeptide antibodies do not substantiallycross react (i.e. does not react specifically) with a protein which is,for example, less than 85% homologous to any of SEQ ID Nos:8-14; e.g.,less than 95% homologous with one of SEQ ID Nos:8-14; e.g., less than98-99% homologous with one of SEQ ID Nos:8-14. By “not substantiallycross react”, it is meant that the antibody has a binding affinity for anon-homologous protein which is at least one order of magnitude, morepreferably at least 2 orders of magnitude, and even more preferably atleast 3 orders of magnitude less than the binding affinity of theantibody for one or more of the proteins of SEQ ID Nos:8-14.

[0174] Following immunization of an animal with an antigenic preparationof a hedgehog protein, anti-hh antisera can be obtained and, if desired,polyclonal anti-hh antibodies isolated from the serum. To producemonoclonal antibodies, antibody-producing cells (lymphocytes) can beharvested from an immunized animal and fused by standard somatic cellfusion procedures with immortalizing cells such as myeloma cells toyield hybridoma cells. Such techniques are well known in the art, aninclude, for example, the hybridoma technique (originally developed byKohler and Milstein, (1975) Nature, 256: 495-497), the human B cellhybridoma technique (Kozbar et al., (1983) Immunology Today, 4: 72), andthe EBV-hybridoma technique to produce human monoclonal antibodies (Coleet al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc. pp. 77-96). Hybridoma cells can be screened immunochemically forproduction of antibodies specifically reactive with a vertebrate hhpolypeptide of the present invention and monoclonal antibodies isolatedfrom a culture comprising such hybridoma cells.

[0175] The term antibody as used herein is intended to include fragmentsthereof which are also specifically reactive with one of the subjectvertebrate hh polypeptides. Antibodies can be fragmented usingconventional techniques and the fragments screened for utility in thesame manner as described above for whole antibodies. For example, F(ab)₂fragments can be generated by treating antibody with pepsin. Theresulting F(ab)₂ fragment can be treated to reduce disulfide bridges toproduce Fab fragments. The antibody of the present invention is furtherintended to include bispecific and chimeric molecules having affinityfor a hedgehog protein conferred by at least one CDR region of theantibody.

[0176] Both monoclonal and polyclonal antibodies (Ab) directed againstauthentic hedgehog polypeptides, or hedgehog variants, and antibodyfragments such as Fab and F(ab)₂, can be used to block the action of oneor more hedgehog proteins and allow the study of the role of theseproteins in, for example, embryogenesis and/or maintenance ofdifferential tissue. For example, purified monoclonal Abs can beinjected directly into the limb buds of chick or mouse embryos. It isdemonstrated in the examples below that hh is expressed in the limb budsof, for example, day 10.5 embryos. Thus, the use of anti-hh Abs duringthis developmental stage can allow assessment of the effect of hh on theformation of limbs in vivo. In a similar approach, hybridomas producinganti-hh monoclonal Abs, or biodegradable gels in which anti-hh Abs aresuspended, can be implanted at a site proximal or within the area atwhich hh action is intended to be blocked. Experiments of this naturecan aid in deciphering the role of this and other factors that may beinvolved in limb patterning and tissue formation.

[0177] Antibodies which specifically bind hedgehog epitopes can also beused in immunohistochemical staining of tissue samples in order toevaluate the abundance and pattern of expression of each of the subjecthh polypeptides. Anti-hedgehog antibodies can be used diagnostically inimmuno-precipitation and immuno-blotting to detect and evaluate hedgehogprotein levels in tissue as part of a clinical testing procedure. Forinstance, such measurements can be useful in predictive valuations ofthe onset or progression of neurological disorders, such as those markedby denervation-like or disuse-like symptoms. Likewise, the ability tomonitor hh levels in an individual can allow determination of theefficacy of a given treatment regimen for an individual afflicted withsuch a disorder. The level of hh polypeptides may be measured in bodilyfluid, such as in samples of cerebral spinal fluid or amniotic fluid, orcan be measured in tissue, such as produced by biopsy. Diagnostic assaysusing anti-hh antibodies can include, for example, imrunoassays designedto aid in early diagnosis of a neurodegenerative disorder, particularlyones which are manifest at birth. Diagnostic assays using anti-hhpolypeptide antibodies can also include immunoassays designed to aid inearly diagnosis and phenotyping of a differentiative disorder, as wellas neoplastic or hyperplastic disorders.

[0178] Another application of anti-hh antibodies of the presentinvention is in the immunological screening of cDNA librariesconstructed in expression vectors such as λgt11, λgt18-23, λZAP, andλORF8. Messenger libraries of this type, having coding sequencesinserted in the correct reading frame and orientation, can producefusion proteins. For instance, λgt11 will produce fusion proteins whoseamino termini consist of β-galactosidase amino acid sequences and whosecarboxy termini consist of a foreign polypeptide. Antigenic epitopes ofan hh protein, e.g. other orthologs of a particular hedgehog protein orother homologs from the same species, can then be detected withantibodies, as, for example, reacting nitrocellulose filters lifted frominfected plates with anti-hh antibodies. Positive phage detected by thisassay can then be isolated from the infected plate. Thus, the presenceof hedgehog homologs can be detected and cloned from other animals, ascan alternate isoforms (including splicing variants) from humans.

[0179] Moreover, the nucleotide sequences determined from the cloning ofhh genes from vertebrate organisms will further allow for the generationof probes and primers designed for use in identifying and/or cloninghedgehog homologs in other cell types, e.g. from other tissues, as wellas hh homologs from other vertebrate organisms. For instance, thepresent invention also provides a probe/primer comprising asubstantially purified oligonucleotide, which oligonucleotide comprisesa region of nucleotide sequence that hybridizes under stringentconditions to at least 10 consecutive nucleotides of sense or anti-sensesequence selected from the group consisting of SEQ ID No:1, SEQ ID No:2,SEQ ID No:3, SEQ ID No:4, SEQ ID No:5, SEQ ID No:6 and SEQ ID No:7, ornaturally occurring mutants thereof. For instance, primers based on thenucleic acid represented in SEQ ID Nos:1-7 can be used in PCR reactionsto clone hedgehog homologs. Likewise, probes based on the subjecthedgehog sequences can be used to detect transcripts or genomicsequences encoding the same or homologous proteins. In preferredembodiments, the probe further comprises a label group attached theretoand able to be detected, e.g. the label group is selected from the groupconsisting of radioisotopes, fluorescent compounds, enzymes, and enzymeco-factors.

[0180] Such probes can also be used as a part of a diagnostic test kitfor identifying cells or tissue which misexpress a hedgehog protein,such as by measuring a level of a hedgehog encoding nucleic acid in asample of cells from a patient; e.g. detecting hh mRNA levels ordetermining whether a genomic hh gene has been mutated or deleted.

[0181] To illustrate, nucleotide probes can be generated from thesubject hedgehog genes which facilitate histological screening of intacttissue and tissue samples for the presence (or absence) ofhedgehog-encoding transcripts. Similar to the diagnostic uses ofanti-hedgehog antibodies, the use of probes directed to hh messages, orto genomic hh sequences, can be used for both predictive and therapeuticevaluation of allelic mutations which might be manifest in, for example,neoplastic or hyperplastic disorders (e.g. unwanted cell growth) orabnormal differentiation of tissue. Used in conjunction withimmunoassays as described above, the oligonucleotide probes can helpfacilitate the determination of the molecular basis for a developmentaldisorder which may involve some abnormality associated with expression(or lack thereof) of a hedgehog protein. For instance, variation inpolypeptide synthesis can be differentiated from a mutation in a codingsequence.

[0182] Accordingly, the present method provides a method for determiningif a subject is at risk for a disorder characterized by aberrant controlof differentiation or unwanted cell proliferation. For instance, thesubject assay can be used in the screening and diagnosis of genetic andacquired disorders which involve alteration in one or more of thehedgehog genes. In preferred embodiments, the subject method can begenerally characterized as comprising: detecting, in a tissue sample ofthe subject (e.g. a human patient), the presence or absence of a geneticlesion characterized by at least one of (i) a mutation of a geneencoding a hedgehog protein or (ii) the mis-expression of a hedgehoggene. To illustrate, such genetic lesions can be detected byascertaining the existence of at least one of (i) a deletion of one ormore nucleotides from a hedgehog gene, (ii) an addition of one or morenucleotides to a hedgehog gene, (iii) a substitution of one or morenucleotides of a hedgehog gene, (iv) a gross chromosomal rearrangementof a hedgehog gene, (v) a gross alteration in the level of a messengerRNA transcript of an hh gene, (vi) the presence of a non-wild typesplicing pattern of a messenger RNA transcript of a vertebrate hh gene,and (vii) a non-wild type level of a hedgehog protein. In one aspect ofthe invention there is provided a probe/primer comprising anoligonucleotide containing a region of nucleotide sequence which iscapable of hybridizing to a sense or antisense sequence selected fromthe group consisting of SEQ ID Nos:1-7, or naturally occurring mutantsthereof, or 5′ or 3′ flanking sequences or intronic sequences naturallyassociated with a vertebrate hh gene. The probe is exposed to nucleicacid of a tissue sample; and the hybridization of the probe to thesample nucleic acid is detected. In certain embodiments, detection ofthe lesion comprises utilizing the probe/primer in a polymerase chainreaction (PCR) (see, e.g., U.S. Pat. Nos: 4,683,195 and 4,683,202) or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al. (1988) Science, 241:1077-1080; and NaKazawa et al. (1944) PNAS91:360-364) the later of which can be particularly useful for detectingpoint mutations in hedgehog genes. Alternatively, immunoassays can beemployed to determine the level of hh proteins, either soluble ormembrane bound.

[0183] Yet another diagnostic screen employs a source of hedgehogprotein directly. As described herein, hedgehog proteins of the presentinvention are involved in the induction of differentiation. Accordingly,the pathology of certain differentiative and/or proliferative disorderscan be marked by loss of hedgehog sensitivity by the afflicted tissue.Consequently, the response of a tissue or cell sample to an inductiveamount of a hedgehog protein can be used to detect and characterizecertain cellular transformations and degenerative conditions. Forinstance, tissue/cell samples from a patient can be treated with ahedgehog agonist and the response of the tissue to the treatmentdetermined. Response can be qualified and/or quantified, for example, onthe basis of phenotypic change as result of hedgehog induction. Forexample, expression of gene products induced by hedgehog treatment canbe scored for by immunoassay. The patched protein, for example, isupregulated in drosophila in response to Dros-HH, and, in light of thefindings herein, a presumed vertebrate homolog will similarly beupregulated. Thus, detection of patched expression on the cells of thepatient sample can permit detection of tissue that is nothedgehog-responsive. Likewise, scoring for other phenotypic markersprovides a means for determining the response to hedgehog.

[0184] Furthermore, by making available purified and recombinanthedgehog polypeptides, the present invention facilitates the developmentof assays which can be used to screen for drugs, including hedgehoghomologs, which are either agonists or antagonists of the normalcellular function of the subject hedgehog polypeptides, or of their rolein the pathogenesis of cellular differentiation and/or proliferation anddisorders related thereto. In one embodiment, the assay evaluates theability of a compound to modulate binding between a hedgehog polypeptideand a hedgehog receptor. A variety of assay formats will suffice and, inlight of the present inventions, will be comprehended by skilledartisan.

[0185] In many drug screening programs which test libraries of compoundsand natural extracts, high throughput assays are desirable in order tomaximize the number of compounds surveyed in a given period of time.Assays which are performed in cell-free systems, such as may be derivedwith purified or semi-purified proteins, are often preferred as“primary” screens in that they can be generated to permit rapiddevelopment and relatively easy detection of an alteration in amolecular target which is mediated by a test compound. Moreover, theeffects of cellular toxicity and/or bioavailability of the test compoundcan be generally ignored in the in vitro system, the assay instead beingfocused primarily on the effect of the drug on the molecular target asmay be manifest in an alteration of binding affinity with receptorproteins. Accordingly, in an exemplary screening assay of the presentinvention, the compound of interest is contacted with a hedgehogreceptor polypeptide which is ordinarily capable of binding a hedgehogprotein. To the mixture of the compound and receptor is then added acomposition containing a hedgehog polypeptide. Detection andquantification of receptor/hedgehog complexes provides a means fordetermining the compound's efficacy at inhibiting (or potentiating)complex formation between the receptor protein and the hedgehogpolypeptide. The efficacy of the compound can be assessed by generatingdose response curves from data obtained using various concentrations ofthe test compound. Moreover, a control assay can also be performed toprovide a baseline for comparison. In the control assay, isolated andpurified hedgehog polypeptide is added to a composition containing thereceptor protein, and the formation of receptor/hedgehog complex isquantitated in the absence of the test compound.

[0186] In an illustrative embodiment, the polypeptide utilized as ahedgehog receptor can be generated from the drosophila patched proteinor a vertebrate homolog thereof. In light of the ability of, forexample, Shh to activate HH pathways in transgenic drosophila (seeExample 4), it may be concluded that vertebrate hedgehog proteins arecapable of binding to drosophila HH receptors. Accordingly, an exemplaryscreening assay includes a suitable portion of the patched protein (SEQID No. 42), such as one or both of the substantial extracellular domains(e.g. residues Lys-93 to His-426 and Arg-700 to Arg-966). For instance,the patched protein can be provided in soluble form, as for example apreparation of one of the extracellular domains, or a preparation ofboth of the extracellular domains which are covalently connected by anunstructured linker (see, for example, Huston et al. (1988) PNAS85:4879; and U.S. Pat. No. 5,091,513), or can be provided as part of aliposomal preparation or expressed on the surface of a cell.

[0187] Complex formation between the hedgehog polypeptide and a hedgehogreceptor may be detected by a variety of techniques. For instance,modulation of the formation of complexes can be quantitated using, forexample, detectably labelled proteins such as radiolabelled,fluorescently labelled, or enzymatically labelled hedgehog polypeptides,by immunoassay, or by chromatographic detection.

[0188] Typically, it will be desirable to immobilize either the hedgehogreceptor or the hedgehog polypeptide to facilitate separation ofreceptor/hedgehog complexes from uncomplexed forms of one of theproteins, as well as to accommodate automation of the assay. In oneembodiment, a fusion protein can be provided which adds a domain thatallows the protein to be bound to a matrix. For example,glutathione-S-transferase/receptor (GST/receptor) fusion proteins can beadsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis,Mo.) or glutathione derivatized microtitre plates, which are thencombined with the hedgehog polypeptide, e.g. an ³⁵S-labeled hedgehogpolypeptide, and the test compound and incubated under conditionsconducive to complex formation, e.g. at physiological conditions forsalt and pH, though slightly more stringent conditions may be desired.Following incubation, the beads are washed to remove any unboundhedgehog polypeptide, and the matrix bead-bound radiolabel determineddirectly (e.g. beads placed in scintillant), or in the supernatant afterthe receptor/hedgehog complexes are dissociated. Alternatively, thecomplexes can dissociated from the bead, separated by SDS-PAGE gel, andthe level of hedgehog polypeptide found in the bead fraction quantitatedfrom the gel using standard electrophoretic techniques.

[0189] Other techniques for immobilizing proteins on matrices are alsoavailable for use in the subject assay. For instance, soluble portionsof the hedgehog receptor protein can be immobilized utilizingconjugation of biotin and streptavidin. For instance, biotinylatedreceptor molecules can be prepared from biotin-NHS(N-hydroxy-succinimide) using techniques well known in the art (e.g.,biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized inthe wells of streptavidin-coated 96 well plates (Pierce Chemical).Alternatively, antibodies reactive with the hedgehog receptor but whichdo not interfere with hedgehog binding can be derivatized to the wellsof the plate, and the receptor trapped in the wells by antibodyconjugation. As above, preparations of a hedgehog polypeptide and a testcompound are incubated in the receptor-presenting wells of the plate,and the amount of receptor/hedgehog complex trapped in the well can bequantitated. Exemplary methods for detecting such complexes, in additionto those described above for the GST-immobilized complexes, includeimmunodetection of complexes using antibodies reactive with the hedgehogpolypeptide, or which are reactive with the receptor protein and competefor binding with the hedgehog polypeptide; as well as enzyme-linkedassays which rely on detecting an enzymatic activity associated with thehedgehog polypeptide. In the instance of the latter, the enzyme can bechemically conjugated or provided as a fusion protein with the hedgehogpolypeptide. To illustrate, the hedgehog polypeptide can be chemicallycross-linked or genetically fused with alkaline phosphatase, and theamount of hedgehog polypeptide trapped in the complex can be assessedwith a chromogenic substrate of the enzyme, e.g.paranitrophenylphosphate. Likewise, a fusion protein comprising thehedgehog polypeptide and glutathione-S-transferase can be provided, andcomplex formation quantitated by detecting the GST activity using1-chloro-2,4-dinitrobenzene (Habig et al (1974) J Biol Chem 249:7130).

[0190] For processes which rely on immunodetection for quantitating oneof the proteins trapped in the complex, antibodies against the protein,such as the anti-hedgehog antibodies described herein, can be used,Alternatively, the protein to be detected in the complex can be “epitopetagged” in the form of a fusion protein which includes, in addition tothe hedgehog polypeptide or hedgehog receptor sequence, a secondpolypeptide for which antibodies are readily available (e.g. fromcommercial sources). For instance, the GST fusion proteins describedabove can also be used for quantification of binding using antibodiesagainst the GST moiety. Other useful epitope tags include myc-epitopes(e.g., see Ellison et al. (1991) J Biol Chem 266:21150-21157) whichincludes a 10-residue sequence from c-myc, as well as the pFLAG system(International Biotechnologies, Inc.) or the pEZZ-protein A system(Pharamacia, N.J.).

[0191] Where the desired portion of the hh receptor (or other hedgehogbinding molecule) cannot be provided in soluble form, liposomal vesiclescan be used to provide manipulatable and isolatable sources of thereceptor. For example, both authentic and recombinant forms of thepatched protein can be reconstituted in artificial lipid vesicles (e.g.phosphatidylcholine liposomes) or in cell membrane-derived vesicles(see, for example, Bear et al. (1992) Cell 68:809-818; Newton et al.(1983) Biochemistry 22:6110-6117; and Reber et al. (1987) J Biol Chem262:11369-11374).

[0192] In addition to cell-free assays, such as described above, thereadily available source of vertebrate hedgehog proteins provided by thepresent invention also facilitates the generation of cell-based assaysfor identifying small molecule agonists/antagonists and the like.Analogous to the cell-based assays described above for screeningcombinatorial libraries, cells which are sensitive to hedgehog inductioncan be contacted with a hedgehog protein and a test agent of interest,with the assay scoring for modulation in hedgehog inductive responses bythe target cell in the presence and absence of the test agent. As withthe cell-free assays, agents which produce a statistically significantchange in hedgehog activities (either inhibition or potentiation) can beidentified. In an illustrative embodiment, motor neuron progenitorcells, such as from neural plate explants, can be used as target cells.Treatment of such explanted cells with, for example, Shh causes thecells to differentiate into motor neurons. By detecting theco-expression of the LIM homeodomain protein Islet-1 (Thor et al. (1991)Neuron 7:881-889; Ericson et al. (1992) Science 256:1555-1560) and theimmunoglobulin-like protein SC1 (Tanaka et al. (1984) Dev Biol106:26-37), the ability of a candidate agent to potentiate or inhibitShh induction of motor neuron differentiation can be measured. Thehedgehog protein can be provided as a purified source, or in the form ofcells/tissue which express the protein and which are co-cultured withthe target cells.

[0193] In yet another embodiment, the method of the present inventioncan be used to isolate and clone hedgehog receptors. For example,purified hedgehog proteins of the present invention can be employed toprecipitate hedgehog receptor proteins from cell fractions prepared fromcells which are responsive to a hedgehog protein. For instance, purifiedhedgehog protein can be derivatized with biotin (using, for instance,NHS-Biotin, Pierce Chemical catalog no. 21420G), and the biotinylatedprotein utilized to saturate membrane bound hh receptors. The hedgehogbound receptors can subsequently be adsorbed or immobilized onstreptavidin. If desired, the hedgehog-receptor complex can becross-linked with a chemical cross-linking agent. In such as manner, hhreceptors can be purified, preferably to near homogeneity. The isolatedhh receptor can then be partially digested with, for example, trypsin,and the resulting peptides separated by reverse-phase chromatography.The chromatography fragments are then analyzed by Edman degradation toobtain single sequences for two or more of the proteolytic fragments.From the chemically determined amino acid sequence for each of thesetryptic fragments, a set of oligonucleotide primers can be designed forPCR. These primers can be used to screen both genomic and cDNAlibraries. Similar strategies for cloning receptors have been employed,for example, to obtain the recombinant gene for somatostatin receptors(Eppler et al. (1992) J Biol Chem 267:15603-15612).

[0194] Other techniques for identifying hedgehog receptors by expressioncloning will be evident in light of the present disclosure. Forinstance, purified hh polypeptides can be immobilized in wells of microtitre plates and contacted with, for example, COS cells transfected witha cDNA library (e.g., from tissue expected to be responsive to hedgehoginduction). From this panning assay, cells which express hedgehogreceptor molecules can be isolated on the basis of binding to theimmobilized hedgehog protein. Another cloning system, described in PCTpublications WO 92/06220 of Flanagan and Leder, involves the use of anexpression cloning system whereby a hedgehog receptor is stored on thebasis of binding to a hedgehog/alkaline phosphatase fusion protein (seealso Cheng et al. (1994) Cell 79:157-168)

[0195] Another aspect of the present invention relates to a method ofinducing and/or maintaining a differentiated state, enhancing survival,and/or promoting proliferation of a cell responsive to a vertebratehedgehog protein, by contacting the cells with an hh agonist or an hhantagonist as the circumstances may warrant. For instance, it iscontemplated by the invention that, in light of the present finding ofan apparently broad involvement of hedgehog proteins in the formation ofordered spatial arrangements of differentiated tissues in vertebrates,the subject method could be used to generate and/or maintain an array ofdifferent vertebrate tissue both in vitro and in vivo. The hh agent,whether inductive or anti-inductive, can be, as appropriate, any of thepreparations described above, including isolated polypeptides, genetherapy constructs, antisense molecules, peptidomimetics or agentsidentified in the drug assays provided herein. Moreover, it iscontemplated that, based on the observation of activity of thevertebrate hedgehog proteins in drosophila, hh agents, for purposes oftherapeutic and diagnostic uses, can include the Dros-HH protein andhomologs thereof. Moreover, the source of hedgehog protein can be, inaddition to purified protein or recombinant cells, cells or tissueexplants which naturally produce one or more hedgehog proteins. Forinstance, as described in Example 2, neural tube explants from embryos,particularly floorplate tissue, can provide a source for Shhpolypeptide, which source can be implanted in a patient or otherwiseprovided, as appropriate, for induction or maintenance ofdifferentiation.

[0196] For example, the present method is applicable to cell culturetechniques. In vitro neuronal culture systems have proved to befundamental and indispensable tools for the study of neural development,as well as the identification of neurotrophic factors such as nervegrowth factor (NGF), ciliary trophic factors (CNTF), and brain derivedneurotrophic factor (BDNF). Once a neuronal cell has becometerminally-differentiated it typically will not change to anotherterminally differentiated cell-type. However, neuronal cells cannevertheless readily lose their differentiated state. This is commonlyobserved when they are grown in culture from adult tissue, and when theyform a blastema during regeneration. The present method provides a meansfor ensuring an adequately restrictive environment in order to maintainneuronal cells at various stages of differentiation, and can beemployed, for instance, in cell cultures designed to test the specificactivities of other trophic factors. In such embodiments of the subjectmethod, the cultured cells can be contacted with an hh polypeptide, oran agent identified in the assays described above, in order to induceneuronal differentiation (e.g. of a stem cell), or to maintain theintegrity of a culture of terminally-differentiated neuronal cells bypreventing loss of differentiation. The source of hedgehog protein inthe culture can be derived from, for example, a purified orsemi-purified protein composition added directly to the cell culturemedia, or alternatively, supported and/or released from a polymericdevice which supports the growth of various neuronal cells and which hasbeen doped with the protein. The source of the hedgehog protein can alsobe a cell that is co-cultured with the intended neuronal cell and whichproduces a recombinant hh. Alternatively, the source can be the neuronalcell itself which has been engineered to produce a recombinant hedgehogprotein. In an exemplary embodiment, a naive neuronal cell (e.g. a stemcell) is treated with an hh agonist in order to induce differentiationof the cells into, for example, sensory neurons or, alternatively,motorneurons. Such neuronal cultures can be used as convenient assaysystems as well as sources of implantable cells for therapeutictreatments. For example, hh polypeptides may be useful in establishingand maintaining the olfactory neuron cultures described in U.S. Pat. No.5,318,907 and the like.

[0197] According to the, present invention, large numbers ofnon-tumorigenic neural progenitor cells can be perpetuated in vitro andinduced to differentiate by contact with hedgehog proteins. Generally, amethod is provided comprising the steps of isolating neural progenitorcells from an animal, perpetuating these cells in vitro or in vivo,preferably in the presence of growth factors, and differentiating thesecells into particular neural phenotypes, e.g., neurons and glia, bycontacting the cells with a hedgehog agonist.

[0198] Progenitor cells are thought to be under a tonic inhibitoryinfluence which maintains the progenitors in a suppressed state untiltheir differentiation is required. However, recent techniques have beenprovided which permit these cells to be proliferated, and unlike neuronswhich are terminally differentiated and therefore non-dividing, they canbe produced in unlimited number and are highly suitable fortransplantation into heterologous and autologous hosts withneurodegenerative diseases.

[0199] By “progenitor” it is meant an oligopotent or multipotent stemcell which is able to divide without limit and, under specificconditions, can produce daughter cells which terminally differentiatesuch as into neurons and glia. These cells can be used fortransplantation into a heterologous or autologous host. By heterologousis meant a host other than the animal from which the progenitor cellswere originally derived. By autologous is meant the identical host fromwhich the cells were originally derived.

[0200] Cells can be obtained from embryonic, post-natal, juvenile oradult neural tissue from any animal. By any animal is meant anymulticellular animal which contains nervous tissue. More particularly,is meant any fish, reptile, bird, amphibian or mammal and the like. Themost preferable donors are mammals, especially mice and humans.

[0201] In the case of a heterologous donor animal, the animal may beeuthanized, and the brain and specific area of interest removed using asterile procedure. Brain areas of particular interest include any areafrom which progenitor cells can be obtained which will serve to restorefunction to a degenerated area of the host's brain. These regionsinclude areas of the central nervous system (CNS) including the cerebralcortex, cerebellum, midbrain, brainstem, spinal cord and ventriculartissue, and areas of the peripheral nervous system (PNS) including thecarotid body and the adrenal medulla. More particularly, these areasinclude regions in the basal ganglia, preferably the striatum whichconsists of the caudate and putamen, or various cell groups such as theglobus pallidus, the subthalamic nucleus, the nucleus basalis which isfound to be degenerated in Alzheimer's Disease patients, or thesubstantia nigra pars compacta which is found to be degenerated inParkinson's Disease patients.

[0202] Human heterologous neural progenitor cells may be derived fromfetal tissue obtained from elective abortion, or from a post-natal,juvenile or adult organ donor. Autologous neural tissue can be obtainedby biopsy, or from patients undergoing neurosurgery in which neuraltissue is removed, in particular during epilepsy surgery, and moreparticularly during temporal lobectomies and hippocampalectomies.

[0203] Cells can be obtained from donor tissue by dissociation ofindividual cells from the connecting extracellular matrix of the tissue.Dissociation can be obtained using any known procedure, includingtreatment with enzymes such as trypsin, collagenase and the like, or byusing physical methods of dissociation such as with a blunt instrument.Dissociation of fetal cells can be carried out in tissue culture medium,while a preferable medium for dissociation of juvenile and adult cellsis artificial cerebral spinal fluid (aCSF). Regular aCSF contains 124 mMNaCl, 5 mM KCl, 1.3 mM MgCl₂, 2 mM CaCl₂, 26 mM NaHCO₃, and 10 mMD-glucose. Low Ca²⁺ aCSF contains the same ingredients except for MgCl₂at a concentration of 3.2 mM and CaCl₂ at a concentration of 0.1 mM.

[0204] Dissociated cells can be placed into any known culture mediumcapable of supporting cell growth, including MEM, DMEM, RPMI, F-12, andthe like, containing supplements which are required for cellularmetabolism such as glutamine and other amino acids, vitamins, mineralsand useful proteins such as transferrin and the like. Medium may alsocontain antibiotics to prevent contamination with yeast, bacteria andfungi such as penicillin, streptomycin, gentamicin and the like. In somecases, the medium may contain serum derived from bovine, equine, chickenand the like. A particularly preferable medium for cells is a mixture ofDMEM and F-12.

[0205] Conditions for culturing should be close to physiologicalconditions. The pH of the culture media should be close to physiologicalpH, preferably between pH 6-8, more preferably close to pH 7, even moreparticularly about pH 7.4. Cells should be cultured at a temperatureclose to physiological temperature, preferably between 30° C.-40° C.,more preferably between 32° C.-38° C., and most preferably between 35°C.-37° C.

[0206] Cells can be grown in suspension or on a fixed substrate, butproliferation of the progenitors is preferably done in suspension togenerate large numbers of cells by formation of “neurospheres” (see, forexample, Reynolds et al. (1992) Science 255:1070-1709; and PCTPublications WO93/01275, WO94/09119, WO94/10292, and WO94/16718). In thecase of propagating (or splitting) suspension cells, flasks are shakenwell and the neurospheres allowed to settle on the bottom corner of theflask. The spheres are then transferred to a 50 ml centrifuge tube andcentrifuged at low speed. The medium is aspirated, the cells resuspendedin a small amount of medium with growth factor, and the cellsmechanically dissociated and resuspended in separate aliquots of media.

[0207] Cell suspensions in culture medium are supplemented with anygrowth factor which allows for the proliferation of progenitor cells andseeded in any receptacle capable of sustaining cells, though as set outabove, preferably in culture flasks or roller bottles. Cells typicallyproliferate within 3-4 days in a 37° C. incubator, and proliferation canbe reinitiated at any time after that by dissociation of the cells andresuspension in fresh medium containing growth factors.

[0208] In the absence of substrate, cells lift off the floor of theflask and continue to proliferate in suspension forming a hollow sphereof undifferentiated cells. After approximately 3-10 days in vitro, theproliferating clusters (neurospheres) are fed every 2-7 days, and moreparticularly every 2-4 days by gentle centrifugation and resuspension inmedium containing growth factor.

[0209] After 6-7 days in vitro, individual cells in the neurospheres canbe separated by physical dissociation of the neurospheres with a bluntinstrument, more particularly by triturating the neurospheres with apipette. Single cells from the dissociated neurospheres are suspended inculture medium containing growth factors, and differentiation of thecells can be induced by plating (or resuspending) the cells in thepresence of a hedgehog agonist, and (optionally) any other factorcapable of sustaining differentiation, such as bFGF and the like.

[0210] To further illustrate other uses of hedgehog agonists andantagonists, it is noted that intracerebral grafting has emerged as anadditional approach to central nervous system therapies. For example,one approach to repairing damaged brain tissues involves thetransplantation of cells from fetal or neonatal animals into the adultbrain (Dunnett et al. (1987) J Exp Biol 123:265-289; and Freund et al.(1985) J Neurosci 5:603-616). Fetal neurons from a variety of brainregions can be successfully incorporated into the adult brain, and suchgrafts can alleviate behavioral defects. For example, movement disorderinduced by lesions of dopaminergic projections to the basal ganglia canbe prevented by grafts of embryonic dopaminergic neurons. Complexcognitive functions that are impaired after lesions of the neocortex canalso be partially restored by grafts of embryonic cortical cells. Theuse of hedgehog proteins or mimetics, such as Shh or Dhh, in the culturecan prevent loss of differentiation, or where fetal tissue is used,especially neuronal stem cells, can be used to induce differentiation.

[0211] Stem cells useful in the present invention are generally known.For example, several neural crest cells have been identified, some ofwhich are multipotent and likely represent uncommitted neural crestcells, and others of which can generate only one type of cell, such assensory neurons, and likely represent committed progenitor cells. Therole of hedgehog proteins employed in the present method to culture suchstem cells can be to induce differentiation of the uncommittedprogenitor and thereby give rise to a committed progenitor cell, or tocause further restriction of the developmental fate of a committedprogenitor cell towards becoming a terminally-differentiated neuronalcell. For example, the present method can be used in vitro to induceand/or maintain the differentiation of neural crest cells into glialcells, schwann cells, chromaffin cells, cholinergic sympathetic orparasympathetic neurons, as well as peptidergic and serotonergicneurons. The hedgehog protein can be used alone, or can be used incombination with other neurotrophic factors which act to moreparticularly enhance a particular differentiation fate of the neuronalprogenitor cell. In the later instance, an hh polypeptide might beviewed as ensuring that the treated cell has achieved a particularphenotypic state such that the cell is poised along a certaindevelopmental pathway so as to be properly induced upon contact with asecondary neurotrophic factor. In similar fashion, even relativelyundifferentiated stem cells or primitive neuroblasts can be maintainedin culture and caused to differentiate by treatment with hedgehogagonists. Exemplary primitive cell cultures comprise cells harvestedfrom the neural plate or neural tube of an embryo even before much overtdifferentiation has occurred.

[0212] In addition to the implantation of cells cultured in the presenceof a functional hedgehog activity and other in vitro uses describedabove, yet another aspect of the present invention concerns thetherapeutic application of a hedgehog protein or mimetic to enhancesurvival of neurons and other neuronal cells in both the central nervoussystem and the peripheral nervous system. The ability of hedgehogprotein to regulate neuronal differentiation during development of thenervous system and also presumably in the adult state indicates thatcertain of the hedgehog proteins can be reasonably expected tofacilitate control of adult neurons with regard to maintenance,functional performance, and aging of normal cells; repair andregeneration processes in chemically or mechanically lesioned cells; andprevention of degeneration and premature death which result from loss ofdifferentiation in certain pathological conditions. In light of thisunderstanding, the present invention specifically contemplatesapplications of the subject method to the treatment of (preventionand/or reduction of the severity of) neurological conditions derivingfrom: (i) acute, subacute, or chronic injury to the nervous system,including traumatic injury, chemical injury, vasal injury and deficits(such as the ischemia resulting from stroke), together withinfectious/inflammatory and tumor-induced injury; (ii) aging of thenervous system including Alzheimer's disease; (iii) chronicneurodegenerative diseases of the nervous system, including Parkinson'sdisease, Huntington's chorea, amylotrophic lateral sclerosis and thelike, as well as spinocerebellar degenerations; and (iv) chronicimmunological diseases of the nervous system or affecting the nervoussystem, including multiple sclerosis.

[0213] Many neurological disorders are associated with degeneration ofdiscrete populations of neuronal elements and may be treatable with atherapeutic regimen which includes a hedgehog agonist. For example,Alzheimer's disease is associated with deficits in severalneurotransmitter systems, both those that project to the neocortex andthose that reside with the cortex. For instance, the nucleus basalis inpatients with Alzheimer's disease have been observed to have a profound(75%) loss of neurons compared to age-matched controls. AlthoughAlzheimer's disease is by far the most common form of dementia, severalother disorders can produce dementia. Several of these are degenerativediseases characterized by the death of neurons in various parts of thecentral nervous system, especially the cerebral cortex. However, someforms of dementia are associated with degeneration of the thalmus or thewhite matter underlying the cerebral cortex. Here, the cognitivedysfunction results from the isolation of cortical areas by thedegeneration of efferents and afferents. Huntington's disease involvesthe degeneration of intrastraital and cortical cholinergic neurons andGABAergic neurons. Pick's disease is a severe neuronal degeneration inthe neocortex of the frontal and anterior temporal lobes, sometimesaccompanied by death of neurons in the striatum. Treatment of patientssuffering from such degenerative conditions can include the applicationof hedgehog polypeptides, or agents which mimic their effects, in orderto control, for example, differentiation and apoptotic events which giverise to loss of neurons (e.g. to enhance survival of existing neurons)as well as promote differentiation and repopulation by progenitor cellsin the area affected. In preferred embodiments, a source of a hedgehogagent is stereotactically provided within or proximate the area ofdegeneration. In addition to degenerative-induced dementias, apharmaceutical preparation of one or more of the subject hedgehogproteins can be applied opportunely in the treatment ofneurodegenerative disorders which have manifestations of tremors andinvoluntary movements. Parkinson's disease, for example, primarilyaffects subcortical structures and is characterized by degeneration ofthe nigrostriatal pathway, raphe nuclei, locus cereleus, and the motornucleus of vagus. Ballism is typically associated with damage to thesubthalmic nucleus, often due to acute vascular accident. Also includedare neurogenic and myopathic diseases which ultimately affect thesomatic division of the peripheral nervous system and are manifest asneuromuscular disorders. Examples include chronic atrophies such asamyotrophic lateral sclerosis, Guillain-Barre syndrome and chronicperipheral neuropathy, as well as other diseases which can be manifestas progressive bulbar palsies or spinal muscular atrophies. The presentmethod is amenable to the treatment of disorders of the cerebellum whichresult in hypotonia or ataxia, such as those lesions in the cerebellumwhich produce disorders in the limbs ipsilateral to the lesion. Forinstance, a preparation of a hedgehog homolog can used to treat arestricted form of cerebellar cortical degeneration involving theanterior lobes (vermis and leg areas) such as is common in alcoholicpatients.

[0214] In an illustrative embodiment, the subject method is used totreat amyotrophic lateral sclerosis. ALS is a name given to a complex ofdisorders that comprise upper and lower motor neurons. Patients maypresent with progressive spinal muscular atrophy, progressive bulbarpalsy, primary lateral sclerosis, or a combination of these conditions.The major pathological abnormality is characterized by a selective andprogressive degeneration of the lower motor neurons in the spinal cordand the upper motor neurons in the cerebral cortex. The therapeuticapplication of a hedgehog agonist, particularly Dhh, can be used alone,or in conjunction with other neurotrophic factors such as CNTF, BDNF orNGF to prevent and/or reverse motor neuron degeneration in ALS patients.

[0215] Hedgehog proteins of the present invention can also be used inthe treatment of autonomic disorders of the peripheral nervous system,which include disorders affecting the innervation of smooth muscle andendocrine tissue (such as glandular tissue). For instance, the subjectmethod can be used to treat tachycardia or atrial cardiac arrythmiaswhich may arise from a degenerative condition of the nerves innervatingthe striated muscle of the heart.

[0216] Furthermore, a potential role for certain of the hedgehogproteins, which is apparent from the appended examples, mainly the dataof respecting hedgehog expression in sensory and motor neurons of thehead and trunk (including limb buds), concerns the role of hedgehogproteins in development and maintenance of dendritic processes of axonalneurons. Potential roles for hedgehog proteins consequently includeguidance for axonal projections and the ability to promotedifferentiation and/or maintenance of the innervating cells to theiraxonal processes. Accordingly, compositions comprising hedgehog agonistsor other hedgehog agents described herein, may be employed to support,or alternatively antagonize the survival and reprojection of severaltypes of ganglionic neurons sympathetic and sensory neurons as well asmotor neurons. In particular, such therapeutic compositions may beuseful in treatments designed to rescue, for example, various neuronsfrom lesion-induced death as well as guiding reprojection of theseneurons after such damage. Such diseases include, but are not limitedto, CNS trauma infarction, infection (such as viral infection withvaricella-zoster), metabolic disease, nutritional deficiency, toxicagents (such as cisplatin treatment). Moreover, certain of the hedgehogagents (such as antagonistic form) may be useful in the selectiveablation of sensory neurons, for example, in the treatment of chronicpain syndromes.

[0217] As appropriate, hedgehog agents can be used in nerve prosthesesfor the repair of central and peripheral nerve damage. In particular,where a crushed or severed axon is intubulated by use of a prostheticdevice, hedgehog polypeptides can be added to the prosthetic device toincrease the rate of growth and regeneration of the dendridic processes.Exemplary nerve guidance channels are described in U.S. Pat. Nos.5,092,871 and 4,955,892. Accordingly, a severed axonal process can bedirected toward the nerve ending from which it was severed by aprosthesis nerve guide which contains, e.g. a semi-solid formulationcontaining hedgehog polypeptide or mimetic, or which is derivatizedalong the inner walls with a hedgehog protein.

[0218] In another embodiment, the subject method can be used in thetreatment of neoplastic or hyperplastic transformations such as mayoccur in the central nervous system. For instance, certain of thehedgehog proteins (or hh agonists) which induce differentiation ofneuronal cells can be utilized to cause such transformed cells to becomeeither post-mitotic or apoptotic. Treatment with a hedgehog agent mayfacilitate disruption of autocrine loops, such as TGF-β or PDGFautostimulatory loops, which are believed to be involved in theneoplastic transformation of several neuronal tumors. Hedgehog agonistsmay, therefore, thus be of use in the treatment of, for example,malignant gliomas, medulloblastomas, neuroectodermal tumors, andependymonas.

[0219] Yet another aspect of the present invention concerns theapplication of the discovery that hedgehog proteins are morphogenicsignals involved in other vertebrate organogenic pathways in addition toneuronal differentiation as described above, having apparent roles inother endodermal patterning, as well as both mesodermal and endodermaldifferentiation processes. As described in the Examples below, Shhclearly plays a role in proper limb growth and patterning by initiatingexpression of signaling molecules, including Bmp-2 in the mesoderm andFgf-4 in the ectoderm. Thus, it is contemplated by the invention thatcompositions comprising hedgehog proteins can also be utilized for bothcell culture and therapeutic methods involving generation andmaintenance of non-neuronal tissue.

[0220] In one embodiment, the present invention makes use of thediscovery that hedgehog proteins, such as Shh, are apparently involvedin controlling the development of stem cells responsible for formationof the digestive tract, liver, lungs, and other organs which derive fromthe primitive gut. As described in the Examples below, Shh serves as aninductive signal from the endoderm to the mesoderm, which is critical togut morphogenesis. Therefore, for example, hedgehog agonists can beemployed in the development and maintenance of an artificial liver whichcan have multiple metabolic functions of a normal liver. In an exemplaryembodiment, hedgehog agonists can be used to induce differentiation ofdigestive tube stem cells to form hepatocyte cultures which can be usedto populate extracellular matrices, or which can be encapsulated inbiocompatible polymers, to form both implantable and extracorporealartificial livers.

[0221] In another embodiment, therapeutic compositions of hedgehogagonists can be utilized in conjunction with transplantation of suchartificial livers, as well as embryonic liver structures, to promoteintraperitoneal implantation, vascularization, and in vivodifferentiation and maintenance of the engrafted liver tissue.

[0222] In yet another embodiment, hedgehog agonists can be employedtherapeutically to regulate such organs after physical, chemical orpathological insult. For instance, therapeutic compositions comprisinghedgehog agonists can be utilized in liver repair subsequent to apartial hepatectomy. Similarly, therapeutic compositions containinghedgehog agonists can be used to promote regeneration of lung tissue inthe treatment of emphysema.

[0223] In still another embodiment of the present invention,compositions comprising hedgehog agonists can be used in the in vitrogeneration of skeletal tissue, such as from skeletogenic stem cells, aswell as the in vivo treatment of skeletal tissue deficiencies. Thepresent invention particularly contemplates the use of hedgehog agonistswhich maintain a skeletogenic activity, such as an ability to inducechondrogenesis and/or osteogenesis. By “skeletal tissue deficiency”, itis meant a deficiency in bone or other skeletal connective tissue at anysite where it is desired to restore the bone or connective tissue, nomatter how the deficiency originated, e.g. whether as a result ofsurgical intervention, removal of tumor, ulceration, implant, fracture,or other traumatic or degenerative conditions.

[0224] For instance, the present invention makes available effectivetherapeutic methods and compositions for restoring cartilage function toa connective tissue. Such methods are useful in, for example, the repairof defects or lesions in cartilage tissue which is the result ofdegenerative wear such as that which results in arthritis, as well asother mechanical derangements which may be caused by trauma to thetissue, such as a displacement of torn meniscus tissue, meniscectomy, alaxation of a joint by a torn ligament, malignment of joints, bonefracture, or by hereditary disease. The present reparative method isalso useful for remodeling cartilage matrix, such as in plastic orreconstructive surgery, as well as periodontal surgery. The presentmethod may also be applied to improving a previous reparative procedure,for example, following surgical repair of a meniscus, ligament, orcartilage. Furthermore, it may prevent the onset or exacerbation ofdegenerative disease if applied early enough after trauma.

[0225] In one embodiment of the present invention, the subject methodcomprises treating the afflicted connective tissue with atherapeutically sufficient amount of a hedgehog agonist, particularly anIhh agonist, to generate a cartilage repair response in the connectivetissue by stimulating the differentiation and/or proliferation ofchondrocytes embedded in the tissue. Induction of chondrocytes bytreatment with a hedgehog agonist can subsequently result in thesynthesis of new cartilage matrix by the treated cells. Such connectivetissues as articular cartilage, interarticular cartilage (menisci),costal cartilage (connecting the true ribs and the sternum), ligaments,and tendons are particularly amenable to treatment in reconstructiveand/or regenerative therapies using the subject method. As used herein,regenerative therapies include treatment of degenerative states whichhave progressed to the point of which impairment of the tissue isobviously manifest, as well as preventive treatments of tissue wheredegeneration is in its earliest stages or imminent. The subject methodcan further be used to prevent the spread of mineralisation intofibrotic tissue by maintaining a constant production of new cartilage.

[0226] In an illustrative embodiment, the subject method can be used totreat cartilage of a diarthroidal joint, such as a knee, an ankle, anelbow, a hip, a wrist, a knuckle of either a finger or toe, or atemperomandibular joint. The treatment can be directed to the meniscusof the joint, to the articular cartilage of the joint, or both. Tofurther illustrate, the subject method can be used to treat adegenerative disorder of a knee, such as which might be the result oftraumatic injury (e.g., a sports injury or excessive wear) orosteoarthritis. An injection of a hedgehog agonist into the joint with,for instance, an arthroscopic needle, can be used to treat the afflictedcartilage. In some instances, the injected agent can be in the form of ahydrogel or other slow release vehicle described above in order topermit a more extended and regular contact of the agent with the treatedtissue.

[0227] The present invention further contemplates the use of the subjectmethod in the field of cartilage transplantation and prosthetic devicetherapies. To date, the growth of new cartilage from eithertransplantation of autologous or allogenic cartilage has been largelyunsuccessful. Problems arise, for instance, because the characteristicsof cartilage and fibrocartilage varies between different tissue: such asbetween articular, meniscal cartilage, ligaments, and tendons, betweenthe two ends of the same ligament or tendon, and between the superficialand deep parts of the tissue. The zonal arrangement of these tissues mayreflect a gradual change in mechanical properties, and failure occurswhen implanted tissue, which has not differentiated under thoseconditions, lacks the ability to appropriately respond. For instance,when meniscal cartilage is used to repair anterior cruciate ligaments,the tissue undergoes a metaplasia to pure fibrous tissue. By promotingchondrogenesis, the subject method can be used to particularly addressesthis problem, by causing the implanted cells to become more adaptive tothe new environment and effectively resemble hypertrophic chondrocytesof an earlier developmental stage of the tissue. Thus, the action ofchondrogensis in the implanted tissue, as provided by the subjectmethod, and the mechanical forces on the actively remodeling tissue cansynergize to produce an improved implant more suitable for the newfunction to which it is to be put.

[0228] In similar fashion, the subject method can be applied toenhancing both the generation of prosthetic cartilage devices and totheir implantation. The need for improved treatment has motivatedresearch aimed at creating new cartilage that is based oncollagen-glycosaminoglycan templates (Stone et al. (1990) Clin OrthopRelat Red 252:129), isolated chondrocytes (Grande et al. (1989) J OrthopRes 7:208; and Takigawa et al. (1987) Bone Miner 2:449), andchondrocytes attached to natural or synthetic polymers (Walitani et al.(1989) J Bone Jt Surg 71B:74; Vacanti et al. (1991) Plast Reconstr Surg88:753; von Schroeder et al. (1991) J Biomed Mater Res 25:329; Freed etal. (1993) J Biomed Mater Res 27:11; and the Vacanti et al. U.S. Pat.No. 5,041,138). For example, chondrocytes can be grown in culture onbiodegradable, biocompatible highly porous scaffolds formed frompolymers such as polyglycolic acid, polylactic acid, agarose gel, orother polymers which degrade over time as function of hydrolysis of thepolymer backbone into innocuous monomers. The matrices are designed toallow adequate nutrient and gas exchange to the cells until engraftmentoccurs. The cells can be cultured in vitro until adequate cell volumeand density has developed for the cells to be implanted. One advantageof the matrices is that they can be cast or molded into a desired shapeon an individual basis, so that the final product closely resembles thepatient's own ear or nose (by way of example), or flexible matrices canbe used which allow for manipulation at the time of implantation, as ina joint.

[0229] In one embodiment of the subject method, the implants arecontacted with a hedgehog agonist during the culturing process, such asan Ihh agonist, in order to induce and/or maintain differentiatedchondrocytes in the culture in order as to further stimulate cartilagematrix production within the implant. In such a manner, the culturedcells can be caused to maintain a phenotype typical of a chondrogeniccell (i.e. hypertrophic), and hence continue the population of thematrix and production of cartilage tissue.

[0230] In another embodiment, the implanted device is treated with ahedgehog agonist in order to actively remodel the implanted matrix andto make it more suitable for its intended function. As set out abovewith respect to tissue transplants, the artificial transplants sufferfrom the same deficiency of not being derived in a setting which iscomparable to the actual mechanical environment in which the matrix isimplanted. The activation of the chondrocytes in the matrix by thesubject method can allow the implant to acquire characteristics similarto the tissue for which it is intended to replace.

[0231] In yet another embodiment, the subject method is used to enhanceattachment of prosthetic devices. To illustrate, the subject method canbe used in the implantation of a periodontal prosthesis, wherein thetreatment of the surrounding connective tissue stimulates formation ofperiodontal ligament about the prosthesis, as well as inhibits formationof fibrotic tissue proximate the prosthetic device.

[0232] In still further embodiments, the subject method can be employedfor the generation of bone (osteogenesis) at a site in the animal wheresuch skeletal tissue is deficient. Indian hedgehog is particularlyassociated with the hypertrophic chondrocytes that are ultimatelyreplaced by osteoblasts. For instance, administration of a hedgehogagent of the present invention can be employed as part of a method fortreating bone loss in a subject, e.g. to prevent and/or reverseosteoporosis and other osteopenic disorders, as well as to regulate bonegrowth and maturation. For example, preparations comprising hedgehogagonists can be employed, for example, to induce endochondralossification, at least so far as to facilitate the formation ofcartilaginous tissue precursors to form the “model” for ossification.Therapeutic compositions of hedgehog agonists can be supplemented, ifrequired, with other osteoinductive factors, such as bone growth factors(e.g. TGF-β factors, such as the bone morphogenetic factors BMP-2 andBMP-4, as well as activin), and may also include, or be administered incombination with, an inhibitor of bone resorption such as estrogen,bisphosphonate, sodium fluoride, calcitonin, or tamoxifen, or relatedcompounds. However, it will be appreciated that hedgehog proteins, suchas Ihh and Shh are likely to be upstream of BMPs, e.g. hh treatment willhave the advantage of initiating endogenous expression of BMPs alongwith other factors.

[0233] In yet another embodiment of the present invention, a hedgehogantagonist can be used to inhibit spermatogenesis. Thus, in light of thepresent finding that hedgehog proteins are involved in thedifferentiation and/or proliferation and maintenance of testicular germcells, hedgehog antagonist can be utilized to block the action of anaturally-occurring hedgehog protein. In a preferred embodiment, thehedgehog antagonist inhibits the biological activity of Dhh with respectto spermatogenesis, by competitively binding hedgehog receptors in thetestis. In similar fashion, hedgehog agonists and antagonists arepotentially useful for modulating normal ovarian function.

[0234] The hedgehog protein, or a pharmaceutically acceptable saltthereof, may be conveniently formulated for administration with abiologically acceptable medium, such as water, buffered saline, polyol(for example, glycerol, propylene glycol, liquid polyethylene glycol andthe like) or suitable mixtures thereof. The optimum concentration of theactive ingredient(s) in the chosen medium can be determined empirically,according to procedures well known to medicinal chemists. As usedherein, “biologically acceptable medium” includes any and all solvents,dispersion media, and the like which may be appropriate for the desiredroute of administration of the pharmaceutical preparation. The use ofsuch media for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe activity of the hedgehog protein, its use in the pharmaceuticalpreparation of the invention is contemplated. Suitable vehicles andtheir formulation inclusive of other proteins are described, forexample, in the book Remington's Pharmaceutical Sciences (Remington'sPharmaceutical Sciences. Mack Publishing Company, Easton, Pa., USA1985). These vehicles include injectable “deposit formulations”. Basedon the above, such pharmaceutical formulations include, although notexclusively, solutions or freeze-dried powders of a hedgehog homolog(such as a Shh, Dhh or Mhh) in association with one or morepharmaceutically acceptable vehicles or diluents, and contained inbuffered media at a suitable pH and isosmotic with physiological fluids.For illustrative purposes only and without being limited by the same,possible compositions or formulations which may be prepared in the formof solutions for the treatment of nervous system disorders with ahedgehog protein are given in U.S. Pat. No. 5,218,094. In the case offreeze-dried preparations, supporting excipients such as, but notexclusively, mannitol or glycine may be used and appropriate bufferedsolutions of the desired volume will be provided so as to obtainadequate isotonic buffered solutions of the desired pH. Similarsolutions may also be used for the pharmaceutical compositions of hh inisotonic solutions of the desired volume and include, but notexclusively, the use of buffered saline solutions with phosphate orcitrate at suitable concentrations so as to obtain at all times isotonicpharmaceutical preparations of the desired pH, (for example, neutralpH).

[0235] Pharmaceutical formulations of the present invention can alsoinclude veterinary compositions, e.g., pharmaceutical preparations ofthe hedgehog proteins, or bioactive fragments thereof, suitable forveterinary uses, e.g., for the treatment of live stock or domesticanimals, e.g., dogs.

[0236] Methods of introduction of exogenous hh at the site of treatmentinclude, but are not limited to, intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, oral, intranasal andtopical. In addition, it may be desirable to introduce thepharmaceutical compositions of the invention into the central nervoussystem by any suitable route, including intraventricular and intrathecalinjection. Intraventricular injection may be facilitated by anintraventricular catheter, for example, attached to a reservoir, such asan Ommaya reservoir.

[0237] Methods of introduction may also be provided by rechargeable orbiodegradable devices. Various slow release polymeric devices have beendeveloped and tested in vivo in recent years for the controlled deliveryof drugs, including proteinacious biopharmaceuticals. A variety ofbiocompatible polymers (including hydrogels), including bothbiodegradable and non-degradable polymers, can be used to form animplant for the sustained release of an hh at a particular target site.Such embodiments of the present invention can be used for the deliveryof an exogenously purified hedgehog protein, which has been incorporatedin the polymeric device, or for the delivery of hedgehog produced by acell encapsulated in the polymeric device.

[0238] An essential feature of certain embodiments of the implant can bethe linear release of the hh, which can be achieved through themanipulation of the polymer composition and form. By choice of monomercomposition or polymerization technique, the amount of water, porosityand consequent permeability characteristics can be controlled. Theselection of the shape, size, polymer, and method for implantation canbe determined on an individual basis according to the disorder to betreated and the individual patient response. The generation of suchimplants is generally known in the art. See, for example, ConciseEncylopedia of Medical & Dental Materials, ed. by David Williams (MITPress: Cambridge, Mass., 1990); and the Sabel et al. U.S. Pat. No.4,883,666. In another embodiment of an implant, a source of cellsproducing a hedgehog protein, or a solution of hydogel matrix containingpurified hh, is encapsulated in implantable hollow fibers. Such fiberscan be pre-spun and subsequently loaded with the hedgehog source(Aebischer et al. U.S. Pat. No. 4,892,538; Aebischer et al. U.S. Pat.No. 5,106,627; Hoffman et al. (1990) Expt. Neurobiol. 110:39-44; Jaegeret al. (1990) Prog. Brain Res. 82:41-46; and Aebischer et al. (1991) J.Biomech. Eng. 113:178-183), or can be co-extruded with a polymer whichacts to form a polymeric coat about the hh source (Lim U.S. Pat. No.4,391,909; Sefton U.S. Pat. No. 4,353,888; Sugamori et al. (1989) Trans.Am. Artif. Intern. Organs 35:791-799; Sefton et al. (1987) Biotehnol.Bioeng. 29:1135-1143; and Aebischer et al. (1991) Biomaterials12:50-55).

[0239] In yet another embodiment of the present invention, thepharmaceutical hedgehog protein can be administered as part of acombinatorial therapy with other agents. For example, the combinatorialtherapy can include a hedgehog protein with at least one trophic factor.Exemplary trophic factors include nerve growth factor, cilliaryneurotrophic growth factor, schwanoma-derived growth factor, glialgrowth factor, stiatal-derived neuronotrophic factor, platelet-derivedgrowth factor, and scatter factor (HGF-SF). Antimitogenic agents canalso be used, for example, when proliferation of surrounding glial cellsor astrocytes is undesirable in the regeneration of nerve cells.Examples of such antimitotic agents include cytosine, arabinoside,5-fluorouracil, hydroxyurea, and methotrexate.

[0240] Another aspect of the invention features transgenic non-humananimals which express a heterologous hedgehog gene of the presentinvention, or which have had one or more genomic hedgehog genesdisrupted in at least one of the tissue or cell-types of the animal.Accordingly, the invention features an animal model for developmentaldiseases, which animal has hedgehog allele which is mis-expressed. Forexample, a mouse can be bred which has one or more hh alleles deleted orotherwise rendered inactive. Such a mouse model can then be used tostudy disorders arising from mis-expressed hedgehog genes, as well asfor evaluating potential therapies for similar disorders.

[0241] Another aspect of the present invention concerns transgenicanimals which are comprised of cells (of that animal) which contain atransgene of the present invention and which preferably (thoughoptionally) express an exogenous hedgehog protein in one or more cellsin the animal. A hedgehog transgene can encode the wild-type form of theprotein, or can encode homologs thereof, including both agonists andantagonists, as well as antisense constructs. In preferred embodiments,the expression of the transgene is restricted to specific subsets ofcells, tissues or developmental stages utilizing, for example,cis-acting sequences that control expression in the desired pattern. Inthe present invention, such mosaic expression of a hedgehog protein canbe essential for many forms of lineage analysis and can additionallyprovide a means to assess the effects of, for example, lack of hedgehogexpression which might grossly alter development in small patches oftissue within an otherwise normal embryo. Toward this and,tissue-specific regulatory sequences and conditional regulatorysequences can be used to control expression of the transgene in certainspatial patterns. Moreover, temporal patterns of expression can beprovided by, for example, conditional recombination systems orprokaryotic transcriptional regulatory sequences.

[0242] Genetic techniques which allow for the expression of transgenescan be regulated via site-specific genetic manipulation in vivo areknown to those skilled in the art. For instance, genetic systems areavailable which allow for the regulated expression of a recombinase thatcatalyzes the genetic recombination a target sequence. As used herein,the phrase “target sequence” refers to a nucleotide sequence that isgenetically recombined by a recombinase. The target sequence is flankedby recombinase recognition sequences and is generally either excised orinverted in cells expressing recombinase activity. Recombinase catalyzedrecombination events can be designed such that recombination of thetarget sequence results in either the activation or repression ofexpression of one of the subject hedgehog proteins. For example,excision of a target sequence which interferes with the expression of arecombinant hh gene, such as one which encodes an antagonistic homologor an antisense transcript, can be designed to activate expression ofthat gene. This interference with expression of the protein can resultfrom a variety of mechanisms, such as spatial separation of the hh genefrom the promoter element or an internal stop codon. Moreover, thetransgene can be made wherein the coding sequence of the gene is flankedby recombinase recognition sequences and is initially transfected intocells in a 3′ to 5′ orientation with respect to the promoter element. Insuch an instance, inversion of the target sequence will reorient thesubject gene by placing the 5′ end of the coding sequence in anorientation with respect to the promoter element which allow forpromoter driven transcriptional activation.

[0243] In an illustrative embodiment, either the cre/loxP recombinasesystem of bacteriophage P1 (Lakso et al. (1992) PNAS 89:6232-6236; Orbanet al. (1992) PNAS 89:6861-6865) or the FLP recombinase system ofSaccharomyces cerevisiae (O'Gorman et al. (1991) Science 251:1351-1355;PCT publication WO 92/15694) can be used to generate in vivosite-specific genetic recombination systems. Cre recombinase catalyzesthe site-specific recombination of an intervening target sequencelocated between loxP sequences. loxP sequences are 34 base pairnucleotide repeat sequences to which the Cre recombinase binds and arerequired for Cre recombinase mediated genetic recombination. Theorientation of loxP sequences determines whether the intervening targetsequence is excised or inverted when Cre recombinase is present(Abremski et al. (1984) J. Biol Chem. 259:1509-1514); catalyzing theexcision of the target sequence when the loxP sequences are oriented asdirect repeats and catalyzes inversion of the target sequence when loxPsequences are oriented as inverted repeats.

[0244] Accordingly, genetic recombination of the target sequence isdependent on expression of the Cre recombinase. Expression of therecombinase can be regulated by promoter elements which are subject toregulatory control, e.g., tissue-specific, developmental stage-specific,inducible or repressible by externally added agents. This regulatedcontrol will result in genetic recombination of the target sequence onlyin cells where recombinase expression is mediated by the promoterelement. Thus, the activation expression of a recombinant hedgehogprotein can be regulated via control of recombinase expression.

[0245] Use of the cre/loxP recombinase system to regulate expression ofa recombinant hh protein requires the construction of a transgenicanimal containing transgenes encoding both the Cre recombinase and thesubject protein. Animals containing both the Cre recombinase and arecombinant hedgehog gene can be provided through the construction of“double” transgenic animals. A convenient method for providing suchanimals is to mate two transgenic animals each containing a transgene,e.g., an hh gene and recombinase gene.

[0246] One advantage derived from initially constructing transgenicanimals containing a hedgehog transgene in a recombinase-mediatedexpressible format derives from the likelihood that the subject protein,whether agonistic or antagonistic, can be deleterious upon expression inthe transgenic animal. In such an instance, a founder population, inwhich the subject transgene is silent in all tissues, can be propagatedand maintained. Individuals of this founder population can be crossedwith animals expressing the recombinase in, for example, one or moretissues and/or a desired temporal pattern. Thus, the creation of afounder population in which, for example, an antagonistic hh transgeneis silent will allow the study of progeny from that founder in whichdisruption of hedgehog mediated induction in a particular tissue or atcertain developmental stages would result in, for example, a lethalphenotype.

[0247] Similar conditional transgenes can be provided using prokaryoticpromoter sequences which require prokaryotic proteins to be simultaneousexpressed in order to facilitate expression of the hedgehog transgene.Exemplary promoters and the corresponding trans-activating prokaryoticproteins are given in U.S. Pat. No. 4,833,080.

[0248] Moreover, expression of the conditional transgenes can be inducedby gene therapy-like methods wherein a gene encoding thetrans-activating protein, e.g. a recombinase or a prokaryotic protein,is delivered to the tissue and caused to be expressed, such as in acell-type specific manner. By this method, a hedgehog transgene couldremain silent into adulthood until “turned on” by the introduction ofthe trans-activator.

[0249] In an exemplary embodiment, the “transgenic non-human animals” ofthe invention are produced by introducing transgenes into the germlineof the non-human animal. Embryonic target cells at various developmentalstages can be used to introduce transgenes. Different methods are useddepending on the stage of development of the embryonic target cell. Thezygote is the best target for micro-injection. In the mouse, the malepronucleus reaches the size of approximately 20 micrometers in diameterwhich allows reproducible injection of 1-2pl of DNA solution. The use ofzygotes as a target for gene transfer has a major advantage in that inmost cases the injected DNA will be incorporated into the host genebefore the first cleavage (Brinster et al. (1985) PNAS 82:4438-4442). Asa consequence, all cells of the transgenic non-human animal will carrythe incorporated transgene. This will in general also be reflected inthe efficient transmission of the transgene to offspring of the foundersince 50% of the germ cells will harbor the transgene. Microinjection ofzygotes is the preferred method for incorporating transgenes inpracticing the invention.

[0250] Retroviral infection can also be used to introduce hedgehogtransgenes into a non-human animal. The developing non-human embryo canbe cultured in vitro to the blastocyst stage. During this time, theblastomeres can be targets for retroviral infection (Jaenich, R. (1976)PNAS 73:1260-1264). Efficient infection of the blastomeres is obtainedby enzymatic treatment to remove the zona pellucida (Manipulating theMouse Embryo, Hogan eds. (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, 1986). The viral vector system used to introduce thetransgene is typically a replication-defective retrovirus carrying thetransgene (Jahner et al. (1985) PNAS 82:6927-6931; Van der Putten et al.(1985) PNAS 82:6148-6152). Transfection is easily and efficientlyobtained by culturing the blastomeres on a monolayer of virus-producingcells (Van der Putten, supra; Stewart et al. (1987) EMBO J. 6:383-388).Alternatively, infection can be performed at a later stage. Virus orvirus-producing cells can be injected into the blastocoele (Jahner etal. (1982) Nature 298:623-628). Most of the founders will be mosaic forthe transgene since incorporation occurs only in a subset of the cellswhich formed the transgenic non-human animal. Further, the founder maycontain various retroviral insertions of the transgene at differentpositions in the genome which generally will segregate in the offspring.In addition, it is also possible to introduce transgenes into the germline by intrauterine retroviral infection of the midgestation embryo(Jahner et al. (1982) supra).

[0251] A third type of target cell for transgene introduction is theembryonic stem cell (ES). ES cells are obtained from pre-implantationembryos cultured in vitro and fused with embryos (Evans et al. (1981)Nature 292:154-156; Bradley et al. (1984) Nature 309:255-258; Gossler etal. (1986) PNAS 83: 9065-9069; and Robertson et al. (1986) Nature322:445-448). Transgenes can be efficiently introduced into the ES cellsby DNA transfection or by retrovirus-mediated transduction. Suchtransformed ES cells can thereafter be combined with blastocysts from anon-human animal. The ES cells thereafter colonize the embryo andcontribute to the germ line of the resulting chimeric animal. For reviewsee Jaenisch, R. (1988) Science 240:1468-1474.

[0252] Methods of making hedgehog knock-out or disruption transgenicanimals are also generally known. See, for example, Manipulating theMouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1986). Recombinase dependent knockouts can also be generated, e.g.by homologous recombination to insert recombinase target sequencesflanking portions of an endogenous hh gene, such that tissue specificand/or temporal control of inactivation of a hedgehog allele can becontrolled as above.

Exemplification

[0253] The invention, now being generally described, will be morereadily understood by reference to the following examples, which areincluded merely for purposes of illustration of certain aspects andembodiments of the present invention and are not intended to limit theinvention.

EXAMPLE 1 Cloning and Expression of Chick Sonic Hedgehog

[0254] (i) Experimental Procedures

[0255] Using degenerate PCR primers, vHH5O(SEQ ID No:18), vHH3O(SEQ IDNo:19) and vHH3I (SEQ ID No:20) corresponding to a sequence conservedbetween Drosophila hedgehog (SEQ ID No:34) (Lee, J. J. et al. (1992)Cell 71: 33-50; Mohler, J. et al., (1992) Development 115: 957-971) andmouse Indian hedgehog (Ihh) (SEQ ID No:10), a 220 base pair (bp)fragment was amplified from chicken genomic DNA. From 15 isolates, twodistinct sequences were cloned, pCHA (SEQ ID No:35) and pCHB (SEQ IDNo:36), each highly homologous to mouse Ihh (FIG. 1). A probe made fromisolate pCHA did not detect expression in embryonic tissues. IsolatepCHB, however, detected a 4 kb message in RNA prepared from embryonichead, trunk, or limb bud RNA. This cloned PCR fragment was thereforeused as a probe to screen an unamplified cDNA library prepared fromHamburger Hamilton stage 22 (Hamburger, W. et al., (1951) J. Morph. 88:49-92) limb bud RNA as described below.

[0256] A single 1.6 kilobase (kb) cDNA clone, pHH-2, was selected forcharacterization and was used in all subsequent analyses. The geneencoding for this cDNA was named Sonic Hedgehog (after the Sega computergame cartoon character). Sequencing of the entire cDNA confirmed thepresence of a single long open reading frame potentially encoding for aprotein of 425 amino acids (aa). The clone extends 220 bp upstream ofthe predicted initiator methionine and approximately 70 bp beyond thestop codon. No consensus polyadenylation signal could be identified inthe 3′ untranslated region. A second potential initiator methionineoccurs at amino acid residue 4. The putative translation initiationsignals surrounding both methionines are predicted to be equallyefficient (Kozak, M., (1987) Nuc. Acids Res. 15: 8125-8132). When thepHH-2 Sonic cDNA is used to probe a northern blot of stage 24 embryonicchick RNA, a single mRNA species of approximately 4 kb is detected inboth limb and trunk tissue. The message size was predicted by comparingit to the position of 18S and 28S ribosomal RNA. Hybridized mRNA wasvisualized after a two day exposure to a phosphoscreen. Because theSonic cDNA clone pHH-2 is only 1.6 kb, it is likely to be missingapproximately 2.4 kb of untranslated sequence.

[0257] PCR Cloning

[0258] All standard cloning techniques were performed according toAusubel et. al. (1989), and all enzymes were obtained from BoehringerMannheim Biochemicals. Degenerate oligonucleotides corresponding toamino acid residues 161 to 237 of the Drosophila hedgehog protein (SEQID No:34) (Lee, J. J. et. al., (1992) Cell 71: 33-50) were synthesized.These degenerate oligonucleotides, vHH5O(SEQ ID No:18), vHH3O(SEQ IDNo:19), and vHH3I (SEQ ID No:20) also contained Eco RI, Cla I, and Xba Isites, respectively, on their 5′ ends to facilitate subcloning. Thenucleotide sequence of these oligos is given below: vHH5O:5′-GGAATTCCCAG(CA)GITG(CT)AA(AG)GA(AG)(CA)(AG)I(GCT)IAA-3′ vHH3O:5′-TCATCGATGGACCCA(GA)TC(GA)AAICCIGC(TC)TC-3′ vHH3I:5′-GCTCTAGAGCTCIACIGCIA(GA)IC(GT)IGC-3′

[0259] where I represents inosine. Nested PCR was performed by firstamplifying chicken genomic DNA using the vHH5O and vHH3O primer pair andthen further amplifying that product using the vHH5O and vHH3I primerpair. In each case the reaction conditions were: initial denaturation at93° C. for 2.5 min., followed by 30 cycles of 94° C. for 45 s, 50° C.for 1 min., 72° C. for 1, and a final incubation of 72° C. for 5 min.The 220 bp PCR product was subcloned into pGEM7zf (Promega). Two uniqueclones, pCHA (SEQ ID No:35) and pCHB (SEQ ID No:36) were identified.

[0260] DNA Sequence Analysis

[0261] Nucleotide sequences were determined by the dideoxy chaintermination method (Sanger, F. et al., (1977) Proc. Natl. Acad. Sci. USA74: 5463-5467) using Sequenase v2.0 T7 DNA polymerase (US Biochemicals).5′ and 3′ nested deletions of pHH-2 were generated by using thenucleases Exo III and S1 (Erase a Base, Promega) and individualsubclones sequenced. DNA and amino acid sequences were analyzed usingboth GCG (Devereux, J. et al., (1984) Nuc. Acids Res. 12: 387-394) andDNAstar software. Searches for related sequences were done through theBLAST network service (Altschul, S. F. et al., (1990) J. Mol. Biol. 215:403-410) provided by the National Center for Biotechnology Information.

[0262] Southern Blot Analysis

[0263] Five (5) μg of chick genomic DNA was digested with Eco RI and/orBam HI, fractionated on a 1% agarose gel, and transferred to a nylonmembrane (Genescreen, New England Nuclear). The filters were probed with³²P-labeled hha or hhb at 42° C. in hybridization buffer (0.5% BSA, 500mM NaHPO₄, 7% SDS, 1 mM EDTA, pH 7.2; Church, G. M. et al., (1984) Proc.Natl. Acad. Sci. USA 81: 1991-1995). The blots were washed at 63° C.once in 0.5% bovine serum albumin, 50 mM NaHPO₄ (pH 7.2), 5% SDS, 1 mMEDTA and twice in 40 mM NaHPO₄ (pH 7.2), 1% SDS, 1 mM EDTA, andvisualized on Kodak XAR-5 film.

[0264] Isolation of Chicken Sonic cDNA Clones

[0265] A stage 22 limb bud cDNA library was constructed in λgt10 usingEco RI/NotI linkers. Unamplified phage plaques (10⁶) were transferred tonylon filters (Colony/Plaque screen, NEN) and screened withα³²P-labelled pooled inserts from PCR clones pCHA (SEQ ID No:35) andpCHB (SEQ ID No:36). Hybridization was performed at 42° C. in 50%formamide 2×SSC, 10% dextran sulfate, 1% SDS and washing as described inthe Southern Blot procedure. Eight positive plaques were identified,purified and their cDNA inserts excised with EcoRI and subcloned intopBluescript SK+ (Stratagene). All eight had approximately 1.7 kb insertswith identical restriction patterns. One, pHH-2, was chosen forsequencing and used in all further manipulations.

[0266] Preparation of Digoxigenin-Labeled Riboprobes

[0267] Plasmid pHH-2 was linearized with Hind III and transcribed withT3 RNA polymerase (for antisense probes) or with Bam HI and transcribedwith T7 RNA polymerase according to the manufacturers instructions forthe preparation of non-radioactive digoxigenin transcripts. Followingthe transcription reaction, RNA was precipitated, and resuspended inRNAse-free water.

[0268] Whole Mount In Situ Hybridization

[0269] Whole-mount in situ hybridization was performed using protocolsmodified from Parr, B. A. et al.. (1993) Development 119: 247-261;Sasaki, H. et al. (1993) Development 118: 47-59; Rosen, B. et al. (1993)Trends Genet. 9: 162-167. Embryos from incubated fertile White Leghorneggs (Spafas) were removed from the egg and extra-embryonic membranesdissected in calcium/magnesium-free phosphate-buffered saline (PBS) atroom temperature. Unless otherwise noted, all washes are for fiveminutes at room temperature. Embryos were fixed overnight at 4° C. with4% paraformaldehyde in PBS, washed twice with PBT (PBS with 0.1%Tween-20) at 4° C., and dehydrated through an ascending methanol seriesin PBT (25%, 50%, 75%, 2×100% methanol). Embryos were stored at −20° C.until further use.

[0270] Both pre-limb bud and limb bud stage embryos were rehydratedthrough an descending methanol series followed by two washes in PBT.Limb bud stage embryos were bleached in 6% hydrogen peroxide in PBT,washed three times with PBT, permeabilized with proteinase K(Boehringer, 2 μg/ml) for 15 minutes, washed with 2 mg/ml glycine in PBTfor 10 minutes, and twice with PBT. Pre-limb bud stage embryos werepermealibized (without prior incubation with hydrogen peroxide) by three30 minute washes in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5%Deoxycholate, 0.1% SDS, 1 mM EDTA, 50 mM Tris-HCl, pH 8.0). In allsubsequent steps, pre-limb bud and limb bud stage embryos were treatedequivalently. Embryos were fixed with 4% paraformaldehyde/0.2%gluteraldehyde in PBT, washed four times with PBT, once withpre-hybridization buffer (50% formamide, 5×SSC, 1% SDS, 50 μg/ml totalyeast RNA, 50 μg/ml heparin, pH 4.5), and incubated with freshpre-hybridization buffer for one hour at 70° C. The pre-hybridizationbuffer was then replaced with hybridization buffer (pre-hybridizationbuffer with digoxigenin labeled riboprobe at 1 μg/ml) and incubatedovernight at 70° C.

[0271] Following hybridization, embryos were washed 3×30 minutes at 70°C. with solution 1 (50% formamide, 5×SSC, 1% SDS, pH 4.5), 3×30 minutesat 70° C. with solution 3 (50% formamide, 2×SSC, pH 4.5), and threetimes at room temperature with TBS (Tris-buffered saline with 2 mMlevamisole) containing 0.1% Tween-20. Non-specific binding of antibodywas prevented by preblocking embryos in TBS/0.1% Tween-20 containing 10%heat-inactivated sheep serum for 2.5 hours at room temperature and bypre-incubating anti-digoxigenin Fab alkaline-phosphatase conjugate(Boehringer) in TBS/0.1% Tween-20 containing heat inactivated 1% sheepserum and approximately 0.3% heat inactivated chick embryo powder. Afteran overnight incubation at 4° C. with the pre-adsorbed antibody inTBS/0.1% Tween-20 containing 1% sheep serum, embryos were washed 3×5minutes at room temperature with TBS/0.1% Tween-20, 5×1.5 hour roomtemperature washes with TBS/1% Tween-20, and overnight with TBS/1%Tween-20 at 4° C. The buffer was exchanged by washing 3×10 minutes withNTMT (100 mM NaCl, 100 mM Tris-HCl, 50 mM MgCl2, 0.1% Tween-20, 2 mMlevamisole). The antibody detection reaction was performed by incubatingembryos with detection solution (NTMT with 0.25 mg/ml NBT and 0.13 mg/mlX-Phos). In general, pre-limb bud stage embryos were incubated for 5-15hours and limb bud stage embryos 1-5 hours. After the detection reactionwas deemed complete, embryos were washed twice with NTMT, once with PBT(pH 5.5), postfixed with 4% paraformaldehyde/0.1% gluteraldehyde in PBT,and washed several times with PBT. In some cases embryos were clearedthrough a series of 30%, 50%, 70%, and 80% glycerol in PBT. Wholeembryos were photographed under transmitted light using a Nikon zoomstereo microscope with Kodak Ektar 100 ASA film. Selected embryos wereprocessed for frozen sections by dehydration in 30% sucrose in PBSfollowed by embedding in gelatin and freezing. 25 μm cryostat sectionswere collected on superfrost plus slides (Fisher), rehydrated in PBS,and mounted with gelvatol. Sections were photographed with Nomarskioptics using a Zeiss Axiophot microscope and Kodak Ektar 25 ASA film.

[0272] (ii) Sequence Homolgy Comparison between Chicken Sonic hh andDrosophila hh and other Vertebrate Sonic hh Proteins

[0273] The deduced Sonic amino acid sequence (SEQ ID No:8) is shown andcompared to the Drosophila hedgehog protein (SEQ ID No:34) in FIG. 2.Over the entire open reading frame the two proteins are 48% homologousat the amino acids level. The predicted Drosophila protein extends 62 aabeyond that of Sonic at its amino terminus. This N-terminal extensionprecedes the putative signal peptide (residues 1-26) of the fly protein(SEQ ID No:34), and has been postulated to be removed during processingof the secreted form of Drosophila hedgehog (Lee, J. J. et al., (1992)Cell 71: 33-50). The sequence of residues 1-26 of the Sonic protein (SEQID No:8) matches well with consensus sequences for eukaryotic signalpeptides (Landry, S. J. et al., (1993) Trends. Biochem. Sci. 16:159-163) and is therefore likely to serve that function for Sonic.Furthermore, FIG. 3 shows a hydropathy plot (Kyte, J. et al., (1982) J.Mol. Biol.157: 133-148) indicating that residues 1-26 of the Sonicprotein (SEQ ID No:8) exhibit a high hydrophobic moment in accord withidentified eukaryotic signal peptides. Cleavage of the putative signalsequence should occur C-terminal to residue 26 according to thepredictive method of von Henjie, G. (1986) Nucl. Acid. Res. 11: 1986. Asingle potential N-linked glycosylation site is located at amino acidresidue 282 of the Sonic protein (SEQ ID No:8). The predicted Sonicprotein does not contain any other strong consensus motifs, and is nothomologous to any other proteins outside of the Hedgehog family.

[0274] The mouse (SEQ ID No:11) and zebrafish (SEQ ID No:12) homologs ofSonic have also been isolated. A comparison of these and the Drosophilasequence is shown schematically in FIG. 4. All of the vertebrateproteins have a similar predicted structure: a putative signal peptideat their amino terminus, followed by an extraordinarily similar 182amino acid region (99% identity in chicken versus mouse and 95% identityin chicken versus zebrafish) and a less well conserved carboxy-terminalregion.

[0275] (iii) At Least Three Hedgehog Homologues are Present in theChicken Genome

[0276] Since two distinct PCR products encoding for chicken hedgehogswere amplified from genomic DNA, the total number of genes in thechicken hedgehog family needed to be estimated. The two PCR clones pCHA(SEQ ID No:35) and pCHB (SEQ ID No:36) were used to probe a genomicSouthern blot under moderately stringent conditions as described in theabove Experimental Procedures. The blot was generated by digesting 5 μgof chick chromosomal DNA with EcoRI and BamHI alone and together. Eachprobe reacted most strongly with a distinct restriction fragment. Forexample, the blot probed with pCHA, shows three bands in each of the BamHI lanes, one strong at 6.6 kb and two weak at 3.4 and 2.7 kb. The blotprobed with pCHB, shows the 2.7 kb band as the most intense, while the3.4 and 6.6 kb bands are weaker. A similar variation of intensities canalso be seen in the Bam HI/Eco RI and EcoRI lanes. Exposure times were72 hr. This data indicates that each probe recognizes a distinct chickenhedgehog gene, and that a third as yet uncharacterized chicken hedgehoghomolog exists in the chicken genome.

[0277] (iv) Northern Analysis Defining Sites of Sonic Transcription

[0278] Northern analysis was performed which confirmed that Sonic isexpressed during chick development. The spatial and temporal expressionof Sonic in the chick embryo from gastrulation to early organogenesiswas determined by whole mount in situ hybridization using a riboprobecorresponding to the full-length Sonic cDNA (SEQ ID No:1).

[0279] 20 μg total RNA isolated from stage 24 chick leg buds or bodies(without heads or limbs) was fractionated on a 0.8% agarose formaldehydegel and transferred to a nylon membrane (Hybond N, Amersham). The blotwas probed with the 1.6 kb EcoRI insert from pHH-2. Random-primedα³²P-labelled insert was hybridized at 42° C. hybridization buffer (1%BSA, 500 mM NaHPO₄, 7% SDS, 1 mM EDTA, pH 7.2) and washed at 63° C. oncein 0.5% bovine serum albumin, 50 mM NaHPO₄ (pH 7.2), 5% SDS, 1 mM EDTAand once in 40 mM NaHPO₄ (pH 7.2), 1% SDS, 1 mM EDTA. The image wasvisualized using a phosphoimager (Molecular Dynamics) and photographeddirectly from the video monitor.

[0280] (v) Expression of Sonic During Mid-Gastrulation

[0281] Sonic message is detected in the gastrulating blastoderm at earlystage 4, the earliest stage analyzed. Staining is localized to theanterior end of the primitive streak in a region corresponding toHensen's node. As gastrulation proceeds, the primitive streak elongatesto its maximal cranial-caudal extent, after which Hensen's noderegresses caudally and the primitive streak shortens. At an early pointof node regression, Sonic mRNA can be detected at the node and inmidline cells anterior to the node. By late stage 5, when the node hasmigrated approximately one-third of the length of the fully elongatedprimitive streak, prominent Sonic expression is seen at the node and inthe midline of the embryo, reaching its anterior limit at the developinghead process. Sections at a cranial level show that Sonic mRNA isconfined to invaginated axial mesendoderm, tissue which contributes toforegut and notochord. More caudally, but still anterior to Hensen'snode, staining of axial mesoderm is absent and Sonic expression isconfined to the epiblast. At the node itself, high levels of Sonicmessage are observed in an asymmetric distribution extending to the leftof and posterior to the primitive pit. This asymmetric distribution isconsistently observed (6/6 embryos from stages 5-7) and is alwayslocated to the left of the primitive pit. At the node, and justposterior to the node, Sonic expression is restricted to the epiblastand is not observed in either mesoderm or endoderm. The expression ofSonic in the dorsal epiblast layer without expression in underlyingaxial mesoderm contrasts markedly with later stages where Sonicexpression in underlying mesoderm always precedes midline neural tubeexpression.

[0282] (vi) Expression of Sonic During Head Fold Stages

[0283] During the formation and differentiation of the head process,Sonic mRNA is detected in midline cells of the neural tube, the foregut,and throughout most of the axial mesoderm. At stage 7, Sonic message isreadily detected asymmetrically at the node and in ventral midline cellsanterior to the node. The rostral limit of Sonic expression extends tothe anterior-most portions of the embryo where it is expressed in theforegut and prechordal mesoderm (Adelmann, H. B., (1932) Am. J. Anat.31, 55-101). At stage 8, expression of Sonic persists along the entireventral midline anterior to Hensen's node, while the node region itselfno longer expresses Sonic. Transverse sections at different axial levelsreveal that at stage 8 Sonic is coexpressed in the notochord and theoverlying ventromedial neuroectoderm from anterior to Hensen's node tothe posterior foregut. The levels of Sonic message are not uniform inthe neural tube: highest levels are found at the presumptive mid- andhindbrain regions with progressively lower levels anterior andposterior. The increasing graded expression in the neural tube fromHensen's node to the rostral brain may reflect the developmental age ofthe neuroectoderm as differentiation proceeds from posterior toanterior. At the anterior-most end of the embryo, expression is observedin midline cells of the dorsal and ventral foregut as well as inprechordal mesoderm. Although the prechordal mesoderm is in intimatecontact with the overlying ectoderm, the latter is devoid of Sonicexpression.

[0284] (vii) Expression of Sonic During Early CNS Differentiation

[0285] At stages 10 through 14, Sonic expression is detected in thenotochord, ventral neural tube (including the floor plate), and gutprecursors. By stage 10, there is a marked expansion of the cephalicneuroectoderm, giving rise to the fore- mid- and hind-brain. At stage10, Sonic mRNA is abundantly expressed in the ventral midline of thehindbrain and posterior midbrain. This expression expands laterally inthe anterior midbrain and posterior forebrain. Expression does notextend to the rostral forebrain at this or later stages. Sections revealthat Sonic is expressed in the notochord, the prechordal mesoderm, andthe anterior midline of the foregut. Expression in the neuroepitheliumextends from the forebrain caudally. In the posterior-most regions ofthe embryo which express Sonic, staining is found only in the notochordand not in the overlying neurectoderm. This contrasts with earlierexpression in which the posterior domains of Sonic expression containcells are located in the dorsal epiblast, but not in underlying mesodermor endoderm. Midgut precursors at the level of the anterior intestinalportal also show weak Sonic expression.

[0286] At stage 14, expression continues in all three germ layers. Theepithelium of the closing midgut expresses Sonic along with portions ofthe pharyngeal endoderm and anterior foregut. Ectoderm lateral andposterior to the tail bud also exhibits weak expression. At this stage,Sonic is also expressed along entire length of the notochord which nowextends rostrally only to the midbrain region and no longer contacts theneuroepithelium at the anterior end of the embryo. Expression in headmesenchyme anterior to the notochord is no longer observed. In theneural tube Sonic is found along the ventral midline of the fore- mid-and hindbrain and posteriorly in the spinal cord. In the forebrain,expression is expanded laterally relative to the hindbrain. At midgutlevels, expression of Sonic in the neural tube appears to extend beyondthe floor plate into more lateral regions. As observed at stage 10,Sonic at stage 14 is found in the notochord, but not in the ventralneural tube in posterior-most regions of the embryo. Whenneuroectodermal expression is first observed in the posterior embryo, itis located in midline cells which appear to be in contact with thenotochord. At later stages, expression continues in areas which showexpression at stage 14, namely the CNS, gut epithelium including theallantoic stalk, and axial mesoderm.

[0287] (viii) Sonic is Expressed in Posterior Limb Bud Mesenchyme

[0288] The limb buds initially form as local thickenings of the lateralplate mesoderm. As distal outgrowth occurs during stage 17, Sonicexpression becomes apparent in posterior regions of both the forelimband the hindlimb. Sections through a stage 21 embryo at the level of theforelimbs reveal that expression of Sonic in limb buds is limited tomesenchymal tissue. A more detailed expression profile of Sonic duringlimb development is discussed below in Example 3. Briefly, as the limbbud grows out, expression of Sonic narrows along the anterior-posterioraxis to become a thin stripe along the posterior margin closely apposedto the ectoderm. Expression is not found at more proximal regions of thebud. High levels of Sonic expression are maintained until around stage25/26 when staining becomes weaker. Expression of Sonic is no longerobserved in wing buds or leg buds after stage 28.

EXAMPLE 2 Mouse Sonic Hedgehog is Implicated in the Regulation of CNSand Limb Polarity

[0289] (i) Experimental Procedures

[0290] Isolation Of Hedgehog Phage Clones

[0291] The initial screen for mammalian hh genes was performed, asabove, using a 700 bp PCR fragment encompassing exons 1 and 2 of theDrosophila hh gene. Approximately one million plaques of a 129/Sv LambdaFix II genomic library (Stratagene) were hybridized with an α ³²P-dATPlabeled probe at low stringency (55° C. in 6×SSC, 0.5%SDS, 5×Denhardt's;final wash at 60° C. in 0.5×SSC, 0.1% SDS for 20′). Five crosshybridizing phage plaques corresponding to the Dhh gene were purified.Restriction enzyme analysis indicated that all clones were overlapping.Selected restriction enzyme digests were then performed to map andsubclone one of these. Subclones in pGEM (Promega) or Bluescript(Stratagene) which cross-hybridized with the Drosophila hh fragmentwhere sequenced using an ABI automatic DNA sequencer.

[0292] Mouse Ihh and Shh were identified by low stringency hybridization(as described above) with a chick Shh cDNA clone to one million plaquesof an 8.5 day λgt10 mouse embryo cDNA library (Fahiner, K. et al.,(1987) EMBO J. 6: 1265-1271). Phage plaques containing a 1.8 kb Ihh and0.64 and 2.8 kb Shh inserts were identified. Inserts were excised andsubcloned into Bluescript (Stratagene) for dideoxy chain terminationsequencing using modified T7 DNA polymerase (USB). The larger Shh clonecontained a partially processed cDNA in which intron splicing at theexon 1/2 junction had not occurred.

[0293] To screen for additional Ihh and Shh cDNA clones, an 8.5 dayλZAPII cDNA library was probed at high stringency (at 65° C. in 6×SSC,0.5% SDS, 5×Denhardt's; final wash at 65° C. in 0.1×SSC, 0.1% SDS for30′) with the Ihh and Shh mouse cDNA clones. No additional Ihh cloneswere identified. However several 2.6 kb, apparently fill length, Shhclones were isolated. The DNA sequence of the additional 5′ codingregion not present in the original 0.64 and 2.8 kb Shh clones wasobtained by analysis of one of the 2.6 kb inserts.

[0294] Northern Blot Analysis

[0295] Expression of Shh was investigated by RNA blot analysis using 20μg of total RNA from adult brain, spleen, kidney, liver, lung, 16.5 dpcbrain, liver and lung; 9.5 dpc to 17.5 dpc whole embryo; 9.5 dpcforebrain, rnidbrain and 10.5 dpc brain. RNA samples wereelectrophoretically separated on a 1.2% agarose gel, transferred andu.v. crosslinked to Genescreen (DuPont) and probed with 2×10⁶ cpm/ml ofan α³²P-dATP labeled mouse Shh probe (2.8 kb insert from λgt 10 screen).Hybridization was performed at 42° C. in 50% formamide 5×Denhardt×s,5×SSPE, 0.1%SDS, 6.5% dextran, 200 μg/ml salmon sperm DNA. Final washwas at 55° C. in 0.1×SSC, 0.1%SDS. The blot was exposed for 6 days inthe presence of an intensifying screen.

[0296] In Situ Hybridization, β-Galactosidase Staining and HistologicalAnalysis

[0297] Embryos from 7.25 to 14.5 dpc were analyzed for either Shh orHNF-3β expression by whole mount in situ hybridization to digoxygeninlabeled RNA probes as described in Wilkinson, (1992) In situHybridization: A Practical Approach. Oxford; Parr et al., (1993)Development 119:247-261. The mouse Shh probe was either a 2.8 kb or 0.6kb RNA transcript generated by T7 (2.8 kb) or T3 (0.6 kb) transcriptionof XbaI and HindIII digests of Bluescript (Stratagene) subclones of theoriginal Shh cDNA inserts. The HNF-3β probe was generated by HindIIIlinearization of a HNF-3β cDNA clone (Sasaki, H. et al., (1993)Development 118: 47-59) and T7 polymerase transcription of 1.6 kbtranscript. Embryos were photographed on an Olympus-SZH photomicroscopeusing Kodak Ektachrome EPY 64T color slide film.

[0298] Sections through wild type and WEXP2-CShh transgenic embryos wereprepared and hybridized with ³⁵S-UIP labeled RNA probes (Wilkinson, D.G. et al., (1987) Development 99: 493-500). Sections were photographedas described in McMahon, A. P. et al., (1992) Cell 69: 581-595.

[0299] β Staining of WEXP2-lacZ embryos with βwas performed according toWhiting, J. et al., (1991) Genes & Dev. 5: 2048-2059. Generalhistological analysis of wildtype and WEXP2-CShh transgenic embryos wasperformed on paraffin sections of Bouin's fixed embryos counterstainedwith haematoxylin and eosin. Histological procedures were as describedby Kaufman, M. H. (1992) The Atlas of Mouse Development, London:Academic Press. Sections were photographed on a Leitz Aristoplancompound microscope using Kodak EPY 64T color slide film.

[0300] DNA Constructs for Transgenics

[0301] Genomic Wnt-l fragments were obtained by screening a λGEM12(Promega) 129/Sv mouse genomic library with a 375 bp MluI-BglII fragmentderived from the fourth exon of the murine Wnt-l gene. One of the clones(W1-15.1) was used in this study.

[0302] As an initial step towards the generation of the pWEXP2expression vector, W1-15.1 was digested to completion with restrictionenzymes AatII and ClaI, and a 2774 bp AatII-ClaI fragment isolated. Thisfragment was ligated into AatII and ClaI cut pGEM-7Zf vector (Promega),generating pW1-18. This plasmid was digested with HindII and ligated toannealed oligonucleotides lacl (SEQ ID No:21) and lac2 (SEQ ID No:22)generating pW1-18S* which has a modified polylinker downstream of theClaI restriction site. This construct pW1-18S*) was digested with ClaIand BglII and ligated with both the 2.5 kb 3′ ClaI-BglII exon-intronregion and 5.5 kb 3′ BglII-BglII Wnt-1 enhancer, generating pWRES4. Thisconstruct contains a 10.5 kb genomic region which starts upstream of theWnt-1 translation initiation codon (at an AatII site approximately 1.0kb from the ATG) and extends to a BglII site 5.5 kb downstream of theWnt-l polyadenylation signal. This plasmid also contains a 250 bp regionof the neomycin phosphotransferase (neo) gene inserted in inverseorientation in the 3′ transcribed but untranslated region. Finally, togenerate the WEXP2 expression vector, a 2 kb Sfi I fragment wasamplified from pWRES4 using Sf-1 (SEQ ID No:23) and Sf-2 (SEQ ID No:24)oligonucleotides. This amplified fragment was digested with Sfi I andinserted into Sfi I linearised pWRES4, generating pWEXP2. This destroysthe Wnt-l translation initiation codon, and replaces it by a polylinkercontaining Nru I, Eco RV, Sac II, and Bst BI restriction sites, whichare unique in pWBXP2.

[0303] The WEXP2-lacZ construct was obtained by inserting an end-filledBgl II-Xho I lacZ fragment isolated from the pSDKlacZpA vector in theNru I cut pWEXP2 expression vector. Similarly, the WEXP2-CShh constructwas obtained by inserting an end-filled XbaI cDNA fragment containingthe full Chick Shh coding sequence (SEQ ID No:1) into the Nru I cutWEXP2 expression vector. Oligonucleotide sequences are as follows: lac1:5′-AGCTGTCGACGCGGCCGCTACGTAGGTTACCGACGTCAAGCTTAGATCTC-3′ lac2:5′-AGCTGAGATCTAAGCTTGACGTCGGTAACCTACGTAGCGGCCGCGTCGAC-3′ Sf-1:5′-GATGGGCCAGGCAGGCCTCGCGATATCGTCACCGCGGTATTCGAA-3′ Sf-2:5′-AGTGCCAGTCGGGGCCCCCAGGGCCGGGCC-3′

[0304] Production and Genotyping of Transgenic Embryos

[0305] Transgenic mouse embryos were generated by microinjection oflinear DNA fragments into the male pronucleus of B6CBAF1/J(C57BL/6J×CBA/J) zygotes. CD-1 or B6CBAF1/J females were used asrecipients for injected embryos. G_(O) mice embryos were collected at9.5, 10.5, and 11.5 dpc, photographed using an Olympus SZHstereophoto-microscope on Kodak EPY-64T color slide film, then processedas described earlier.

[0306] WEXP2-lacZ and WEXP2-CShh transgenic embryos were identified byPCR analysis of proteinase-K digests of yolk sacs. Briefly, yolk sacswere carefully dissected free from maternal and embryonic tissues,avoiding cross-contamination between littermates, then washed once inPBS. After overnight incubation at 55° C. in 50 μl of PCR proteinase-Kdigestion buffer (McMahon, A. P. et al., (1990) Cell 62: 1073-1085). 1μl of heat-inactivated digest was subjected to polymerase chain reaction(PCR) in a 20 μl volume for 40 cycles as follows: 94° C. for 30 seconds,55° C. for 30 seconds, 72° C. for 1 minute, with the reactioningredients described previously (McMahon, A. P. et al., (1990) Cell 62:1073-1085)). In the case of the WEXP2-lacZ transgenic embryos,oligonucleotides 137 (SEQ ID No:25) and 138 (SEQ ID No:26) amplify a 352bp lacZ specific product. In the case of the WEXP2-CShh embryos,oligonucleotides WPR2 (Wnt-1-specific) (SEQ ID No:27) and 924 (ChickShh-specific) (SEQ ID No:28) amplify a 345 bp fragment spanning theinsertion junction of the Chick-Shh cDNA in the WEXP2 expression vector.Table 2 summarizes the results of WEXP2-C-Shh transgenic studies.Oligonucleotide sequences are as follows: 137:5′-TACCACAGCGGATGGTTCGG-3′ 138: 5′-GTGGTGGTTATGCCGATCGC-3′ WPR2:5′-TAAGAGGCCTATAAGAGGCGG-3′ 924: 5′-AAGTCAGCCCAGAGGAGACT-3′

[0307] (ii) Mouse hh Genes

[0308] The combined screening of mouse genomic and 8.5 day post coitum(dpc) cDNA libraries identified three mammalian hh counterparts (FIG.5A) which herein will be referred to as Desert, Indian and Sonichedgehog (Dhh, Ihh and Shh, respectively). Sequences encoding Dhh (SEQID No:2) were determined from analysis of clones identified by lowstringency screening of a mouse genomic library. DNA sequencing of oneof five overlapping lambda phage clones identified three homologousregions encoding a single open reading frame interrupted by introns inidentical position to those of the Drosophila hh gene (FIG. 5A).Splicing across the exon 1/2 boundary was confirmed by polymerase chainreaction (PCR) amplification of first strand cDNA generated from adulttesticular RNA. The partial sequence of Ihh (SEQ ID No:3) and thecomplete sequence of Shh (SEQ ID No:4) coding regions were determinedfrom the analysis of overlapping cDNA clones isolated from 8.5 dpc cDNAlibraries. The longest Shh clone, 2.6 kb, appears to be full length whencompared with the Shh transcript present in embryonic RNAs. The 1.8 kbpartial length Ihh cDNA is complete at the 3′ end, as evidenced by thepresence of a polyadenylation consensus sequence and short poly A tail.

[0309] Alignment of the predicted Drosophila hh protein sequence (SEQ IDNo:34) with those of the mouse Dhh (SEQ ID No:9), Ihh (SEQ ID No:10) andShh (SEQ ID No:11), and chick Shh (SEQ ID No:8) and zebrafish Shh (SEQID No:12), reveals several interesting features of the hh-family (FIG.5A). All the vertebrate hh-proteins contain an amino terminalhydrophobic region of approximately 20 amino acids immediatelydownstream of the initiation methionine. Although the properties ofthese new hh proteins have not been investigated, it is likely that thisregion constitutes a signal peptide and vertebrate hhs are secretedproteins. Signal peptide cleavage is predicted to occur (von Heijne, G.,(1986) Nucleic Acids Research 14: 4683-4690) just before an absolutelyconserved six amino acid stretch, CGPGRG (SEQ ID No:29) (correspondingto residues 85-90)(FIG. 5A), in all hh proteins. This generatesprocessed mouse Dhh (SEQ ID No:9) and Shh (SEQ ID No:11) proteins of 41and 44 kd, respectively. Interestingly, Drosophila hh (SEQ ID No:34) ispredicted to contain a substantial amino terminal extension beyond thehydrophobic domain suggesting that the Drosophila protein enters thesecretory pathway by a type II secretory mechanism. This would generatea transmembrane tethered protein which would require subsequent cleavageto release a 43 kd secreted form of the protein. In vitro analysis ofDrosophila hh is consistent with this interpretation (Lee, J. J. et al.,(1992) Cell 71: 33-50). However, there also appears to be transitionalinitiation at a second methionine (position 51 of SEQ ID No:34) justupstream of the hydrophobic region (Lee, J. J. et al., (1992) Cell 71:33-50), suggesting that Drosophila hh, like its vertebrate counterparts,may also be secreted by recognition of a conventional amino terminalsignal peptide sequence.

[0310] Data base searches for protein sequences related to vertebratehh's failed to identify any significant homologies, excepting Drosophilahh. In addition, searching the “PROSITE” data bank of protein motifs didnot reveal any peptide motifs which are conserved in the different hhproteins. Thus, the hhs represent a novel family of putative cellsignaling molecules.

[0311] One feature of the amino acid alignment is the high conservationof hh sequences. Vertebrate hhs share 47 to 51% amino acid identity withDrosophila hh throughout the predicted processed polypeptide sequence(FIG. 6). Dhh has a slightly higher identity than that of Ihh and Shhsuggesting that Dhh may be the orthologue of Drosophila hh. Conservationis highest in the amino terminal half of the proteins, indeed, fromposition 85 (immediately after the predicted shared cleavage site) to249, 62% of the amino acids are completely invariant amongst theDrosophila and vertebrate proteins. Comparison of mouse Dhh, Ihh and Shhwhere their sequences overlap in this more conserved region, indicatesthat Ihh and Shh are more closely related (90% amino acid identity;residues 85 to 266) than with the Dhh sequence (80% amino acid identity;residues 85 to 266). Thus, Ihh and Shh presumably resulted from a morerecent gene duplication event.

[0312] Comparison of cross species identity amongst Shh proteins revealsan even more striking sequence conservation. Throughout the entirepredicted processed sequence mouse and chick Shh share 84% of amino acidresidues (FIG. 6). However, in the amino terminal half (positions 85 to266) mouse and chick are 99% and mouse and zebrafish 94% identical in an180 amino acid stretch. Conservation falls off rapidly after position266 (FIG. 5A). SEQ ID No:40 shows the consensus sequence in the aminoterminal half of all vertebrate Shh genes (human, mouse, chicken andzebrafish) identified to date. SEQ ID No:41 shows the consensus sequencein the amino terminal half of vertebrate hedgehog genes (Shh, Ihh, andDhh) identified to date in different species (mouse, chicken, human andzebrafish).

[0313] In summary, hh family members are likely secreted proteinsconsisting of a highly conserved amino terminal and more divergentcarboxyl terminal halves. The extreme interspecies conservation of thevertebrate Shh protein points to likely conservation of Shh functionacross vertebrate species.

[0314] (iii) Expression of Mouse Shh at the Axial Midline

[0315] Expression of Shh in the mouse was examined in order to explorethe role of mouse Shh (SEQ ID No:11) in vertebrate development. Northernblots of embryonic and adult RNA samples were probed with aradiolabelled mouse Shh CDNA probe. An Shh transcript of approximately2.6 kb was detected in 9.5 dpc whole embryo RNA, and 9.5 and 10.5 dpcbrain RNA fractions. No expression was detected in total RNA samplesfrom later embryonic stages. Of the late fetal and adult tissue RNAsexamined Shh expression was only detected in 16.5 dpc and adult lung.

[0316] To better define the precise temporal and spatial expression ofShh an extensive series of whole mount and serial section in situhybridizations were performed using digoxygenin and ³⁵S-radiolabelledRNA probes, respectively, to mouse embryo samples from 7.25 dpc (midstreak egg cylinder stage of gastrulation) to 13.5 dpc. No Shhexpression is detected at mid-gastrulation stages (7.25 dpc) prior tothe appearance of the node, the mouse counterpart of the amphibianorganizer and chick Hensen's node. When the primitive streak is fullyextended and the midline mesoderm of the head process is emerging fromthe node (7.5 to 7.75 dpc), Shh is expressed exclusively in the headprocess. At late head fold stages, Shh is expressed in the node andmidline mesoderm of the head process extending anteriorly under thepresumptive brain. Just prior to somite formation, Shh extends to theanterior limit of the midline mesoderm, underlying the presumptivemidbrain. As somites are formed, the embryonic axis extends caudally.The notochord, which represents the caudal extension of the headprocess, also expresses Shh, and expression is maintained in the node.

[0317] Interestingly, by 8 somites (8.5 dpc) strong Shh expressionappears in the CNS. Expression is initiated at the ventral midline ofthe midbrain, above the rostral limit of the head process. By 10 somitesCNS expression in the midline extends rostrally in the forebrain andcaudally into the hindbrain and rostral spinal cord. Expression isrestricted in the hindbrain to the presumptive floorplate, whereasmidbrain expression extends ventro-laterally. In the forebrain, there isno morphological floor plate, however ventral Shh expression here iscontinuous with the midbrain. By 15 somites ventral CNS expression iscontinuous from the rostral limit of the diencephalon to the presumptivespinal cord in somitic regions. Over the next 18 to 24 hrs, to the 25-29somite stage, CNS expression intensifies and forebrain expressionextends rostral to the optic stalks. In contrast to all other CNSregions, in the rostral half of the diencephalon, Shh is not expressedat the ventral midline but in two strips immediately lateral to thisarea which merge again in the floor of the forebrain at its rostrallimit. Expression of Shh in both the notochord and floorplate isretained until at least 13.5 dpc.

[0318] Several groups have recently reported the cloning and expressionof vertebrate members of a family of transcription factors, related tothe Drosophila forkhead gene. One of these, HNF-3β shows severalsimilarities in expression to Shh (Sasaki, H. et al., (1993) Development118: 47-59) suggesting that HNF-3β may be a potential regulator of Shh.To investigate this possibility, direct comparison of HNF-3β and Shhexpression was undertaken. HNF-3β transcripts are first detected in thenode (as previously reported by Sasaki, H. et al., (1993) supra), priorto the emergence of the head process and before Shh is expressed. Fromthe node, expression proceeds anteriorly in the head process, similar toShh expression. Activation of HNF-3β within the CNS is first observed at2-3 somites, in the presumptive mid and hindbrain, prior to the onset ofShh expression. By 5 somites, expression in the midbrain broadensventro-laterally, extends anteriorly into the forebrain and caudally inthe presumptive floor plate down much of the neuraxis in the somiticregion. Strong expression is maintained at this time in the node andnotochord. However, by 10 somites expression in the head process is lostand by 25-29 somites notochordal expression is only present in the mostextreme caudal notochord. In contrast to the transient expression ofHNF-3β in the midline mesoderm, expression in the floor plate is stablyretained until at least 11.5 dpc. Thus, there are several spatialsimilarities between the expression of HNF-3β and Shh in both themidline mesoderm and ventral CNS and it is likely that both genes areexpressed in the same cells. However, in both regions, HNF-3β expressionprecedes that of Shh. The main differences are in the transientexpression of HNF-3β in the head process and notochord and Shhexpression in the forebrain. Whereas HNF-3β and Shh share a similarbroad ventral and ventral lateral midbrain and caudal diencephalicexpression, only Shh extends more rostrally into the forebrain. Ingeneral, these results are consistent with a model in which initialactivation of Shh expression may be regulated by HNF-3β.

[0319] The similarity in Shh and HNF-3β expression domains is alsoapparent in the definitive endoderm which also lies at the midline.Broad HNF-3β expression in the foregut pocket is apparent at 5 somitesas previously reported by Sasaki, H. et al., (1993) supra. Shh is alsoexpressed in the endoderm, immediately beneath the forebrain. Both genesare active in the rostral and caudal endoderm from 8 to 11 somites.Whereas HNF-3β is uniformly expressed, Shh expression is initiallyrestricted to two ventro-lateral strips of cells. Ventral restrictedexpression of Shh is retained in the most caudal region of thepresumptive gut until at least 9.5 dpc whereas BNF-3β is uniformlyexpressed along the dorso-ventral axis. Both genes are expressed in thepharyngeal ectoderm at 9.5 dpc and expression is maintained in the gutuntil at least 11.5 dpc. Moreover, expression of Shh in the embryonicand adult lung RNA suggests that endodermal expression of Shh maycontinue in, at least some endoderm derived organs.

[0320] (iv) Expression of Shh in the Limb

[0321] Expression of Shh is not confined to midline structures. By 30-35somites (9.75 dpc), expression is detected in a small group of posteriorcells in the forelimb bud. The forelimb buds form as mesenchymaloutpocketings on the flanks, opposite somites 8 to 12, at approximatelythe 17 to 20 somite stage. Shh expression is not detectable in theforelimbs until about 30-35 somites, over 12 hours after the initialappearance of the limbs. Expression is exclusively posterior andrestricted to mesenchymal cells. By 10.5 dpc, both the fore andhindlimbs have elongated substantially from the body flank. At this timeShh is strongly expressed in the posterior, distal aspect of both limbsin close association with the overlying ectoderm. Analysis of sectionsat this stage detects Shh expression in an approximately six cell widestrip of posterior mesenchymal cells. In the forelimb, Shh expressionceases by 11.5 dpc. However, posterior, distal expression is stilldetected in the hindlimb. No limb expression is detected beyond 12.5dpc.

[0322] (v) Ectopic Expression of Shh

[0323] Grafting studies carried out principally in the chick demonstratethat cell signals derived from the notochord and floor plate pattern theventral aspect of the CNS (as described above). In the limb, a transientsignal produced by a group of posterior cells in both limb buds, thezone of polarizing activity (ZPA), is thought to regulate patterningacross the anterior-posterior axis. Thus, the sequence of Shh, whichpredicts a secreted protein and the expression profile in midlinemesoderm, the floor plate and in the limb, suggest that Shh signalingmay mediate pattern regulation in the ventral CNS and limb.

[0324] To determine whether Shh may regulate ventral development in theearly mammalian CNS, a Wnt-l enhancer was used to alter its normaldomain of expression. Wnt-l shows a dynamic pattern of expression whichis initiated in the presumptive midbrain just prior to somite formation.As the neural folds elevate and fuse to enclose the neural tube, Wnt-1expression in the midbrain becomes restricted to a tight circle, justanterior of the midbrain, the ventral midbrain and the dorsal midline ofthe diencephalon, midbrain, myelencephalon and spinal cord (Wilkinson,D. G. et al., (1987) Cell 50: 79-88; McMahon, A. P. et al., (1992) Cell69: 581-595; Parr, B. A. et al., (1993) Development 119: 247-261).

[0325] It was determined that essentially normal expression of lacZreporter constructs within the Wnt-l expression domain is dependent upona 5.5 kb enhancer region which lies downstream of the Wnt-1polyadenylation sequence. A construct was generated for ectopicexpression of cDNA clones in the Wnt-l domain and tested in transgenicsusing a lacZ reporter (pWEXP-lacZ; FIG. 9). Two of the four G_(O)transgenic embryos showed readily detectable β-galactosidase activity,and in both expression occurred throughout the normal Wnt-l expressiondomain. More extensive studies with a similar construct also containingthe 5.5 kb enhancer gave similar frequencies. Some ectopic expressionwas seen in newly emerging neural crest cells, probably as a result ofperdurance of β-galactosidase RNA or protein in the dorsally derivedcrest. Thus, the Wnt-l expression construct allows the efficient ectopicexpression of cDNA sequences in the midbrain and in the dorsal aspect ofmuch of the CNS.

[0326] An Shh ectopic expression construct (pWEXP-CShh) containing twotandem head to tail copies of a chick Shh cDNA was generated (FIG. 7).By utilizing this approach, ectopic expression of the chick Shh isdistinguishable from that of the endogenous mouse Shh gene. Chick Shhshows a high degree of sequence identity and similar expression to themouse gene. Thus, it is highly likely that Shh function is widelyconserved amongst vertebrates, a conclusion further supported by studiesof the same gene in zebrafish.

[0327] Table 2 shows the results of several transgenic experiments inwhich the G_(O) population was collected at 9.5 to 11.5 dpc.Approximately half of the transgenic embryos identified at each stage ofdevelopment had a clear, consistent CNS phenotype. As we expect, on thebasis of control studies using the 5.5 kb Wnt-l enhancer, that only halfthe transgenics will express the transgene, it is clear that in mostembryos ectopically expressing chick Shh, an abnormal phenotype results.TABLE 2 Summary of WEXP2-Chick Shh transgenic studies Number of Numberof Number of Embryos with Age (dpc) Embryos Transgenics CNSphenotype^(a)  9.5 37 11 6 (54.5%) 10.5 59 16 8 (50%)   11.5 33  7 3(42.9%)

[0328] At 9.5 dpc, embryos with a weaker phenotype show an open neuralplate from the mid diencephalon to the myelencephalon. In embryos with astronger phenotype at the same stage, the entire diencephalon is openand telencephalic and optic development is morphologically abnormal. Asthe most anterior diencephalic expression of Wnt-l is lower than that inmore caudal regions, the differences in severity may relate todifferences in the level of chick Shh expression in different G_(O)embryos. At the lateral margins of the open neural folds, where Wnt-l isnormally expressed, there is a thickening of the neural tissue extendingfrom the diencephalon to myelencephalon. The cranial phenotype issimilar at 10.5 and 11.5 dpc. However, there appears to be a retardationin cranial expansion of the CNS at later stages.

[0329] In addition to the dorsal cranial phenotype, there is aprogressive dorsal phenotype in the spinal cord. At 9.5 dpc, the spinalcord appears morphologically normal, except at extreme rostral levels.However by 10.5 dpc, there is a dorsal dysmorphology extending to thefore or hindlimbs. By 11.5 dpc, all transgenic embryos showed a dorsalphenotype along almost the entire spinal cord. Superficially, the spinalcord had a rippled, undulating appearance suggestive of a change in cellproperties dorsally. This dorsal phenotype, and the cranial phenotypewere examined by histological analysis of transgenic embryos.

[0330] Sections through a 9.5 dpc embryo with an extreme CNS phenotypeshow a widespread dorsal perturbation in cranial CNS development. Theneural/ectodermal junction in the diencephalon is abnormal. Neuraltissue, which has a columnar epithelial morphology quite distinct fromthe squamous epithelium of the surface ectoderm, appears to spreaddorsolaterally. The myelencephalon, like the diencephalon and midbrain,is open rostrally. Interestingly, there are discontinuous dorso-lateralregions in the myelencephalon with a morphology distinct from the normalroof plate regions close to the normal site of Wnt-l expression. Thesecells form a tight, polarized epithelium with basely located nuclei, amorphology similar to the floor plate and distinct from other CNSregions. Differentiation of dorsally derived neural crest occurs intransgenic embryos as can be seen from the presence of cranial ganglia.In the rostral spinal cord, the neural tube appeared distendeddorso-laterally which may account for the superficial dysmorphology.

[0331] By 11.5 dpc, CNS development is highly abnormal along the entiredorsal spinal cord to the hindlimb level. The dorsal half of the spinalcord is enlarged and distended. Dorsal sensory innervation occurs,however, the neuronal trajectories are highly disorganized. Mostobviously, the morphology of dorsal cells in the spinal cord, whichnormally are elongated cells with distinct lightly staining nuclei andcytoplasm, is dramatically altered. Most of the dorsal half of thespinal cord consists of small tightly packed cells with darkly stainingnuclei and little cytoplasm. Moreover, there appears to be many more ofthese densely packed cells, leading to abnormal outgrowth of the dorsalCNS. In contrast, ventral development is normal, as are dorsal rootganglia, whose origins lie in neural cells derived from the dorsalspinal cord.

[0332] (vi) Ectopic Shh Expression Activates Floor Plate Gene Expression

[0333] To determine whether ectopic expression of chick Shh results ininappropriate activation of a ventral midline development in the dorsalCNS, expression of two floor plate expressed genes, HNF-3β and mouseShh, were examined. Whole mounts of 9.5 dpc transgenic embryos showectopic expression of HNF-3β throughout the cranial Wnt-l expressiondomain. In addition to normal expression at the ventral midline, HNF-3βtranscripts are expressed at high levels, in a circle just rostral tothe mid/hindbrain junction, along the dorsal (actually lateral inunfused brain folds) aspects of the midbrain and, more weakly, in theroof plate of the myelencephalon. No expression is observed in themetencephalon which does not express Wnt-l. Thus, ectopic expression ofShh leads to the activation of HNF-3β throughout the cranial Wnt-lexpression domain.

[0334] The relationship between chick Shh expression and the expressionof HNF-3β in serial sections was also examined. Activation of HNF-3β inthe brain at 9.5 and 10.5 dpc is localized to the dorsal aspect in goodagreement with the observed ectopic expression of chick Shh.Interestingly mouse Shh is also activated dorsally. Thus, two earlyfloor plate markers are induced in response to chick Shh.

[0335] From 9.5 dpc to 11.5 dpc, the spinal cord phenotype becomes moresevere. The possibility that activation of a floor plate pathway mayplay a role in the observed phenotype was investigated. In contrast tothe brain, where ectopic HNF-3β and Shh transcripts are still present,little or no induction of these floor plate markers is observed. Thus,although the dorsal spinal cord shows a widespread transformation incellular phenotype, this does not appear to result from the induction offloor plate development.

EXAMPLE 3 Chick Sonic Hedgehog Mediates ZPA Activity

[0336] (i) Experimental Procedures

[0337] Retinoic Acid Bead Implants

[0338] Fertilized white Leghorn chicken eggs were incubated to stage 20and then implanted with AG1-X2 ion exchange beads (Biorad) soaked in 1mg/ml retinoic acid (RA, Sigma) as described by Tickle, C. et al.,(1985) Dev. Biol 109: 82-95. Briefly, the beads were soaked for 15 minin lmg/ml RA in DMSO, washed twice and implanted under the AER on theanterior margin of the limb bud. After 24 or 36 hours, some of theimplanted embryos were harvested and fixed overnight in 4%paraformaldehyde in PBS and then processed for whole mount in situanalysis as previously described. The remainder of the animals wereallowed to develop to embryonic day 10 to confirm that the dose of RAused was capable of inducing mirror image duplications. Control animalswere implanted with DMSO soaked beads and showed no abnormal phenotypeor gene expression.

[0339] Plasmids

[0340] Unless otherwise noted, all standard cloning techniques wereperformed according to Ausubel, F. M. et al., (1989) Current Protocolsin Molecular Biology (N.Y.: Greene Publishing Assoc. and WileyInerscience), and all enzymes were obtained from Boehringer MannheimBiochemicals. pHH-2 is a cDNA contain the entire coding region ofchicken Sonic hedgehog (SEQ ID No:1). RCASBP(A) and RCASBP(E) arereplication-competent retroviral vectors which encode viruses withdiffering host ranges. RCANBP(A) is a variant of RCASBP(A) from whichthe second splice acceptor has been removed. This results in a viruswhich can not express the inserted gene and acts as a control for theeffects of viral infection (Hughes, S. H. et al., (1987) J. Virol. 61:3004-3012; Fekete, D. et al., (1993) Mol. Cell. Biol. 13: 2604-2613).RCASBP/AP(E) is version of RCASBP(E) containing a human placentalalkaline phosphatase cDNA (Fekete, D. et al., (1993b) Proc. Natl. Acad.Sci. USA 90: 2350-2354). SLAX13 is a pBluescript SK+ derived plasmidwith a second Cla I restriction site and the 5′ untranslated region ofv-src (from the adaptor plasmid CLA12-Nco, Hughes, S. H. et al., (1987)J. Virol. 61: 3004-3012) cloned 5′ of the EcoRI (and ClaI) site in thepBluescript polylinker. RCASBP plasmids encoding Sonic from either thefirst (M1) or second (M2) methionine (at position 4) were constructed byfirst shuttling the 1.7 kb Sonic fragment of pHH-2 into SLAX-13 usingoligonucleotides to modify the 5′ end of the cDNA such that either thefirst or second methionine is in frame with the NcoI site of SLAX-13.The amino acid sequence of Sonic is not mutated in these constructs. TheM1 and M2 Sonic ClaI fragments (v-src 5′UTR:Sonic) were each thensubcloned into RCASBP(A), RCANBP(A) and RCASBP(E), generatingSonic/RCAS-A1, Sonic/RCAS-A2, Sonic/RCAN-A1, Sonic/RCAN-A2,Sonic/RCAS-E1 and Sonic/RCAS-E2.

[0341] Chick Embryos, Cell Lines and Virus Production

[0342] All experimental manipulations were performed on standardspecific-pathogen free White Leghorn chick embryos (S-SPF) from closedflocks provided fertilized by SPAFAS (Norwich, Conn.). Eggs wereincubated at 37.5° C. and staged according to Hamburger, V. et al.,(1951) J. Exp. Morph. 88: 49-92. All chick embryo fibroblasts (CEF) wereprovided by C. Cepko. S-SPF embryos and CEFs have previously been shownto be susceptible to RCASBP(A) infection but resistant to RCASBP(E)infection (Fekete, D. et al., (1993b) Proc. Natl. Acad. Sci. USA 90:2350-2354). Line 15b CEFs are susceptible to infection by both RCASBP(A)and (E). These viral host ranges were confirmed in control experiments.CEF cultures were grown and transfected with retroviral vector DNA asdescribed (Morgan, B. A. et al., (1993) Nature 358: 236-239; Fekete, D.et al., (1993b) Proc. Natl. Acad, Sci. USA 90: 2350-2354). All viruseswere harvested and concentrated as previously described (Morgan, B. A.et al., (1993) Nature 358: 236-239; Fekete, D. et al., (1993b) Proc.Natl. Acad. Sci. USA 90: 2350-2354) and had titers of approximately 10⁸cfu/ml.

[0343] Cell Implants

[0344] A single 60 mm dish containing line 15b CEFs which had beeninfected with either RCASBP/AP(E), Sonic/RCAS-E1 or Sonic/RCAS-E2 weregrown to 50-90% confluence, lightly trypsinized and then spun at 1000rpm for 5 min in a clinical centrifuge. The pellet was resuspended in 1ml media, transferred to a microcentrifuge tube and thenmicrocentrifuged for 2 min at 2000 rpm. Following a 30 min incubation at37° C., the pellet was respun for 2 min at 2000 rpm and then lightlystained in media containing 0.01% nile blue sulfate. Pellet fragments ofapproximately 300 μm×100 μm×50 μm were implanted as a wedge to theanterior region of hh stage 19-23 wing buds (as described by Riley, B.B. et al., (1993) Development 118: 95-104). At embryonic day 10, theembryos were harvested, fixed in 4% paraformaldehyde in PBS, stainedwith alcian green, and cleared in methyl salicylate (Tickle, C. et al.,(1985) Dev. Biol 109: 82-95).

[0345] Viral Infections

[0346] Concentrated Sonic/RCAS-A2 or Sonic/RCAN-A2 was injected underthe AER on the anterior margin of stage 20-22 wing buds. At 24 or 36hours post-infection, the embryos were harvested, fixed in 4%paraformaldehyde in PBS and processed for whole mount in situ analysisas previously described.

[0347] (ii) Co-Localization of Sonic Expression and Zpa Activity

[0348] ZPA activity has been carefully mapped both spatially andtemporally within the limb bud (Honig, L. S. et al., (1985) J. Embryol.exp. Morph. 87: 163-174). In these experiments small blocks of limb budtissue from various locations and stages of chick embryogenesis(Hamburger, V et al., (1951) J. Exp. Morph. 88: 49-92) were grafted tothe anterior of host limb buds and the strength of ZPA activity wasquantified according to degrees of digit duplication. Activity is firstweakly detected along the flank prior to limb bud outgrowth. Theactivity first reaches a maximal strength at stage 19 in the proximalposterior margin of the limb bud. By stage 23 the activity extends thefull length of the posterior border of the limb bud. The activity thenshifts distally along the posterior margin so that by stage 25 it is nolonger detectable at the base of the flank. The activity then fadesdistally until it is last detected at stage 29.

[0349] This detailed map of endogenous polarizing activity provided theopportunity to determine the extent of the correlation between thespatial pattern of ZPA activity and Sonic expression over a range ofdevelopmental stages. Whole mount in situ hybridization was used toassay the spatial and temporal pattern of Sonic expression in the limbbud. Sonic expression is not detected until stage 17, at the initiationof limb bud formation, at which time it is weakly observed in a punctatepattern reflecting a patchy expression in a few cells. From that pointonwards the Sonic expression pattern exactly matches the location of theZPA, as determined by Honig, L. S. et al., (1985) J. Embryol. exp.Morph. 87: 163-174, both in position and in intensity of expression.

[0350] (iii) Induction of Sonic Expression by Retinoic Acid

[0351] A source of retinoic acid placed at the anterior margin of thelimb bud will induce ectopic tissue capable causing mirror-imageduplications (Summerbell, D. et al., (1983) In Limb Development andRegeneration (N.Y.: Ala R. Liss) pp. 109-118; Wanek, N. et al., (1991)Nature 350: 81-83). The induction of this activity is not an immediateresponse to retinoic acid but rather takes approximately 18 hours todevelop (Wanek, N. et al., (1991) Nature 350: 81-83). When it doesdevelop, the polarizing activity is not found surrounding the implantedretinoic acid source, but rather is found distal to it in the mesenchymealong the margin of the limb bud (Wanek, N. et al., (1991) Nature 350:81-83).

[0352] If Sonic expression is truly indicative of ZPA tissue, then itshould be induced in the ZPA tissue which is ectopically induced byretinoic acid. To test this, retinoic acid-soaked beads were implantedin the anterior of limb buds and the expression of Sonic after variouslengths of time using whole-mount in situ hybridization was assayed. Asthe limb bud grows, the bead remains imbedded proximally in tissue whichbegins to differentiate. Ectopic Sonic expression is first detected inthe mesenchyme 24 hours after bead implantation. This expression isfound a short distance from the distal edge of the bead. By 36 hoursSonic is strongly expressed distal to the bead in a stripe just underthe anterior ectoderm in a mirror-imnage pattern relative to theendogenous Sonic expression in the posterior of the limb bud.

[0353] (iv) Effects of Ectopic Expression of Sonic on Limb Patterning

[0354] The normal expression pattern of Sonic, as well as that inducedby retinoic acid, is consistent with Sonic being a signal produced bythe ZPA. To determine whether Sonic expression is sufficient for ZPAactivity, the gene was ectopically expressed within the limb bud. Inmost of the experiments we have utilized a variant of areplication-competent retroviral vector called RCAS (Hughes, S. H. etal., (1987) J. Virol. 61: 3004-3012)) both as a vehicle to introduce theSonic sequences into chick cells and to drive their expression. The factthat there exists subtypes of avian retroviruses which have host rangesrestricted to particular strains of chickens was taken advantage of tocontrol the region infected with the Sonic/RCAS virus (Weiss, R. (etal.) (1984) RNA Tumor Viruses, Vol. 1 Weiss et al. eds., (N.Y.: ColdSpring Harbor Laboratories) pp. 209-260); Fekete, D. et al., (1993a)Mol. Cell. Biol. 13: 2604-2613). Thus a vector with a type E envelopeprotein (RCAS-E, Fekete, D. et al., (1993b) Proc. Natl. Acad. Sci. USA90: 2350-2354) is unable to infect the cells of the SPAFAS outbred chickembryos routinely used in our lab. However, RCAS-E is able to infectcells from chick embryos of line 15b. In the majority of experiments,primary chick embryo fibroblasts (CEFs) prepared from line 15b embryosin vitro were infected. The infected cells were pelleted and implantedinto a slit made in the anterior of S-SPF host limb buds. Due to therestricted host range of the vector, the infection was thus restrictedto the graft and did not spread through the host limb bud.

[0355] To determine the fate of cells implanted and to control for anyeffect of the implant procedure, a control RCAS-E vector expressinghuman placental alkaline phosphatase was used. Alkaline phosphataseexpression can be easily monitored histochemically and the location ofinfected cells can thus be conveniently followed at any stage. Within 24hours following implantation the cells are dispersed proximally anddistally within the anterior margin of the limb bud. Subsequently, cellsare seen to disperse throughout the anterior portion of the limb andinto the flank of the embryo.

[0356] Limb buds grafted with alkaline phosphatase expressing cells oruninfected cells give rise to limbs with structures indistinguishablefrom unoperated wild type limbs. Such limbs have the characteristicanterior-to-posterior digit pattern 2-3-4. ZPA grafts give rise to avariety of patterns of digits depending on the placement of the graftwithin the bud (Tickle, C. et al., (1975) Nature 254: 199-202) and theamount of tissue engrafted (Tickle, C. (1981) Nature 289: 295-298). Insome instances the result can be as weak as the duplication of a singledigit 2. However, in optimal cases the ZPA graft evokes the productionof a full mirror image duplication of digits 4-3-2-2-3-4 or 4-3-2-3-4(see FIG. 8). A scoring system has been devised which rates theeffectiveness of polarizing activity on the basis of the most posteriordigit duplicated: any graft which leads to the development of aduplication of digit 4 has been defined as reflecting 100% polarizingactivity (Honig, L. S. et al., (1985) J. Embryol. Exp. Morph.87:163-174).

[0357] Grafts of 15b fibroblasts expressing Sonic resulted in a range ofZPA-like phenotypes. In some instances the resultant limbs deviate fromthe wild type solely by the presence of a mirror-image duplication ofdigit 2. The most common digit phenotype resulting from graftingSonic-infected CEF cells is a mirror-image duplication of digits 4 and 3with digit 2 missing: 4-3-3-4. In many such cases the two central digitsappear fused in a 4-3/3-4 pattern. In a number of the cases the graftsinduced fill mirror-image duplications of the digits equivalent tooptimal ZPA grafts 4-3-2-2-3-4. Besides the digit duplications, theectopic expression of Sonic also gave rise to occasional duplications ofproximal elements including the radius or ulna, the humerus and thecoracoid. While these proximal phenotypes are not features of ZPAgrafts, they are consistent with an anterior-to-posteriorrespecification of cell fate. In some instances, most commonly when theradius or ulna was duplicated, more complex digit patterns wereobserved. Typically, an additional digit 3 was formed distal to aduplicated radius.

[0358] The mirror-image duplications caused by ZPA grafts are notlimited to skeletal elements. For example, feather buds arenormally-present only along the posterior edge of the limb. Limbsexhibiting mirror-image duplications as a result of ectopic Sonicexpression have feather buds on both their anterior and posterior edges,similar to those observed in ZPA grafts.

[0359] While ZPA grafts have a powerful ability to alter limb patternwhen placed at the anterior margin of a limb bud, they have no effectwhen placed at the posterior margin (Saunders, J. W. et al., (1968)Epithelial-Mesenchymal Interaction, Fleischmayer and Billingham, eds.(Baltimore: Williams and Wilkins) pp. 78-97). Presumably, the lack ofposterior effect is a result of polarizing activity already beingpresent in that region of the bud. Consistent with this, grafts of Sonicexpressing cells placed in the posterior of limb buds never result inchanges in the number of digits. Some such grafts did producedistortions in the shape of limb elements, the most common being aslight posterior curvature in the distal tips of digits 3 and 4 whencompared to wild type wings.

[0360] (v) Effect of Ectopic Sonic Expression on Hoxd Gene Activity

[0361] The correct expression of Hoxd genes is part of the process bywhich specific skeletal elements are determined (Morgan, B. A. et al.,(1993) Nature 358: 236-239). A transplant of a ZPA into the anterior ofa chick limb bud ectopically activates sequential transcription of Hoxdgenes in a pattern which mirrors the normal sequence of Hoxd geneexpression (Nohno, T. et al., (1991) Cell 64: 1197-1205;Izpisua-Belmonte, J. C. et al., (1991) Nature 350: 585-589). Sinceectopic Sonic expression leads to the same pattern duplications as a ZPAgraft, we reasoned that Sonic would also lead to sequential activationof Hoxd genes.

[0362] To test this hypothesis, anterior buds were injected withSonic/RCAS-A2, a virus which is capable of directly infecting the hoststrains of chicken embryos. This approach does not strictly limit theregion expressing Sonic (being only moderately controlled by the timing,location and titer of viral injection), and thus might be expected togive a more variable result. However, experiments testing the kineticsof viral spread in infected limb buds indicate that infected cellsremain localized near the anterior margin of the bud for at least 48hours. Hoxd gene expression was monitored at various times postinfection by whole mount in situ hybridization. As expected, these genesare activated in a mirror-image pattern relative their expression in theposterior of control limbs. For example, after 36 hours Hoxd-13 isexpressed in a mirror-image symmetrical pattern in the broadened distalregion of infected limb buds. Similar results were obtained with otherHoxd genes (manuscript in preparation).

EXAMPLE 4 A Functionally Conserved Homolog of Drosophila Hedgehog isExpressed in Tissues with Polarizing Activity in Zebrafish Embryos

[0363] (i) Experimental Procedures

[0364] Cloning and Sequencing

[0365] Approximately 1.5×10⁶ plaques of a 33h zebrafish embryonic λgt11cDNA library were screened by plaque hybridization at low stringency(McGinnis, W. et al., (1984) Nature 308: 428-433) using a mix of two hhsequences as a probe: a Drosophila hh 400 bp EcoRI fragment and a murineIhh 264 bp BamHI-EcoRI exon 2 fragment. Four clones were isolated andsubcloned into the EcoRI sites of pUC18 T3T7 (Pharmacia). Both strandsof clone 8.3 were sequenced using nested deletions (Pharmacia) andinternal oligonucleotide primers. DNA sequences and derived amino acidsequences were analyzed using “Geneworks” (Intelligenetics) and the GCGsoftware packages.

[0366] PCR Amplification

[0367] Degenerate oligonucleotides hh5.1 (SEQ ID No:30) and hh3.3 (SEQID No:31) were used to amplify genomic zebrafish DNA hh 5.1:AG(CA)GJTG(CT)AA(AG)GA(AG)(CA)(AG)I(GCT)IAA hh 3.3:CTCIACIGCIA(GA)ICK=(GT)IGCIA

[0368] PCR was performed with an initial denaturation at 94° C. followedby 35 cycles of 47° C. for 1 min, 72° C. for 2 min and 94° C. for 1 minwith a final extension at 72° C. Products were subdcloned in pUC18(Pharmacia).

[0369] In Situ Hybridization

[0370] In situ hybridizations of zebrafish embryos were performed asdescribed in Oxtoby, E. et al., (1993) Nuc. Acids REs. 21: 1087-1095with the following modifications: Embryos were rehydrated in ethanolrather than methanol series; the proteinase K digestion was reduced to 5min and subsequent washes were done in PBTw without glycine; theantibody was preadsorbed in PBTw, 2 mg/ml BSA without sheep serum; andantibody incubation was performed in PBTw, 2 mg/ml BSA. Drosophilaembryos were processed and hybridized as previously described.

[0371] Histology

[0372] Stained embryos were dehydrated through ethanol:butanol series,as previously described (Godsave, S. F. et al., (1988) Development 102:555-566), and embedded in Fibrowax. 8 μm sections were cut on an Anglianrotary microtome

[0373] RNA Probe Synthesis

[0374] For analysis of Shh expression, two different templates were usedwith consistent results; (i) phh[c] 8.3 linearized with Bg1 II totranscribe an antisense, RNA probe that excludes the conserved region,and (ii) phh[c] 8.3 linearized with Hind III to transcribe an antisenseRNA that covers the complete cDNA. All in situ hybridizations wereperformed with the latter probe which gives better signal. Other probeswere as follows: Axial DraI-linearised p6TlN (Strähle, U. et al., (1993)Genes & Dev. 7: 1436-1446) using T3 RNA polymerase. gsc linearized withEcoR1 and transcribed with T7: pax 2 Bam HI-linearized pcF16 (Krauss, S.et al., (1991) Development 113: 1193-1206) using T7 RNA polymerase. Insitu hybridizations were performed using labelled RNA at a concentrationof 1 ng/ml final concentration. Antisense RNA probes were transcribedaccording to the manufacturer's protocol (DIG RNA Labelling Kit, BCL).

[0375] Zebrafish Strains

[0376] Wild type fish were bred from a founder population obtained fromthe Goldfish Bowl, Oxford. The mutant cyclops strain b16 and the mutantnotail strains b160 and b195 were obtained from Eugene, Oreg. Fish werereared at 28° C. on a 14 h light/10 h dark cycle.

[0377] RNA Injections

[0378] The open reading frame of Shh was amplified by PCR, usingoligonucleotides 5′-CTGCAGGGATCCACCATGCGGCTTTTGACGAG-3′ (SEQ ID No:32),which contains a consensus Kozak sequence for translation initiation,and 5′-CTGCAGGGATCCTTATTCCACACGAGGGATT-3′ (SEQ ID No:33), and subclonedinto the BglII site of pSP64T (Kreig, P. A. et al., (1984) Nuc.AcidsRes. 12: 7057-7070). This vector includes 5′ and 3′ untranslated Xenopusβ-Globin sequences for RNA stabilization and is commonly used for RNAinjections experiments in Xenopus. In vitro transcribed Shh RNA at aconcentration of approximately 100 μg/ml was injected into a single cellof naturally spawned zebrafish embryos at one-cell to 4-cell stagesusing a pressure-pulsed Narishige microinjector. The injected volume waswithin the picolitre range. Embryos were fixed 20 to 27 hrs afterinjection in BT-Fix (Westerfield, M. (1989) The Zebrafish Book, (Eugene:The University of Oregon Press)) and processed as described above forwhole-mount in situ hybridizations with the axial probe.

[0379] Transgenic Drosophila

[0380] An EcoR1 fragment, containing the entire Shh ORF, was purifiedfrom the plasmid phh[c]8.3 and ligated with phosphatased EcoR1 digestedtransformation vector pCaSpeRhs (Thummel, C. S. et al., (1988) Gene 74:445-456). The recombinant plasmid, pHS Shh containing the Shh ORF in thecorrect orientation relative to the heat shock promoter, was selectedfollowing restriction enzyme analysis of miniprep DNA from transformedcolonies and used to transform Drosophila embryos using standardmicroinjection procedures (Roberts, D. B. (1986), Drosophila, APractical Approach, Roberts, D. B., ed., (Oxford: IRL Press) pp. 1-38).

[0381] Ectopic Expression in Drosophila Embryos

[0382] Embryos carrying the appropriate transgenes were collected over 2hr intervals, transferred to thin layers of 1% agarose on glassmicroscope slides and incubated in a plastic Petri dish floating in awater bath at 37° C. for 30 min intervals. Following heat treatment,embryos were returned to 25° C. prior to being fixed for in situhybridization with DIG labelled single stranded Shh, wg or ptc RNAprobes as previously described (Ingham et al., (1991) Curr. Opin. Genet.Dev. 1: 261-267).

[0383] (ii) Molecular Cloning of Zebrafish Hedgehog Homologues

[0384] In an initial attempt to isolate sequences homologous toDrosophila hh, a zebrafish genomic DNA library was screened at reducedstringency with a partial cDNA, hhPCR4.1, corresponding to the first andsecond exons of the Drosophila gene (Mohler, J. et al., (1992)Development 115: 957-971). This screen proved unsuccessful; however, asimilar screen of a mouse genomic library yielded a single clone withsignificant homology to hh., subsequently designated Ihh. A 264 bpBamHI-EcoRI fragment from this lambda clone containing sequenceshomologous to the second exon of the Drosophila gene was subcloned and,together with the Drosophila partial cDNA fragment, used to screen aλgt11 zebrafish cDNA library that was prepared from RNA extracted from33 h old embryos. This screen yielded four clones with overlappinginserts the longest of which is 1.6 kb in length, herein referred to asShh (SEQ ID No:5).

[0385] (iii) A Family of Zebrafish Genes Homologous to the DrosophilaSegment Polarity Gene, Hedgehog

[0386] Alignment of the predicted amino acid sequences of Shh (SEQ IDNo:12) and hh (SEQ ID No:34) revealed an identity of 47%, confirmingthat Shh is a homolog of the Drosophila gene. A striking conservationoccurs within exon 2: an 80 amino acid long domain shows 72% identitybetween Shh and Drosophila hh. (FIG. 9A). This domain is also highlyconserved in all hh-related genes cloned so far and is therefore likelyto be essential to the function of hh proteins. A second domain ofapproximately 30 amino acids close to the carboxy-terminal end, thoughit shows only 61% amino-acid identity, possesses 83% similarity betweenShh and hh when allowing for conservative substitutions and could also,therefore, be of functional importance (FIG. 9B). Although putativesites of post-translational modification can be noted, their position isnot conserved between Shh and hh.

[0387] Lee, J. J. et al., (1992) Cell 71: 33-50, identified ahydrophobic stretch of 21 amino acids flanked downstream by a putativesite of signal sequence cleavage (predicted by the algorithm of vonHeijne, G. (1986) Nuc. Acids Res. 11) close to the amino-terminal end ofhh. Both the hydrophobic stretch and the putative signal sequencecleavage sites of hh, which suggest it to be a signaling molecule, areconserved in Shh. In contrast to hh, Shh does not extend N-terminally tothe hydrophobic stretch.

[0388] Using degenerate oligonucleotides corresponding to amino-acidsflanking the domain of high homology between Drosophila hh and mouse Ihhexons 2 described above, fragments of the expected size were amplifiedfrom zebrafish genomic DNA by PCR. After subcloning and sequencing, itappeared that three different sequences were amplified, all of whichshow high homology to one another and to Drosophila hh (FIG. 10). One ofthese corresponds to Shh therein referred to as 2-hh(a) (SEQ ID No:16)and 2hh(b) (SEQ ID No:17), while the other two represent additionalzebrafish hh homologs (SEQ ID No:5). cDNAs corresponding to one of theseadditional homologs have recently been isolated, confirming that it istranscribed. Therefore, Shh represents a member of a new vertebrate genefamily.

[0389] (iv) Shh Expression in the Developing Zebrafish Embryo

[0390] Gastrula Stages

[0391] Shh expression is first detected at around the 60% epiboly stageof embryogenesis in the dorsal mesoderm. Transcript is present in atriangular shaped area, corresponding to the embryonic shield, theequivalent of the amphibian organizer, and is restricted to the innercell layer, the hypoblast. During gastrulation, presumptive mesodermalcells involute to form the hypoblast, and converge towards the futureaxis of the embryo, reaching the animal pole at approximately 70%epiboly. At this stage, Shh-expressing cells extend over the posteriorthird of the axis, and the signal intensity is not entirely homogeneous,appearing stronger at the base than at the apex of the elongatingtriangle of cells.

[0392] This early spatial distribution of Shh transcript is reminiscentof that previously described for axial, a forkhead-related gene;however, at 80% epiboly, axial expression extends further towards theanimal pole of the embryo and we do not see Shh expression in the headarea at these early developmental stages.

[0393] By 100% epiboly, at 9.5 hours of development, the posterior tipof the Shh expression domain now constitutes a continuous band of cellsthat extends into the head. To determine the precise anterior boundaryof Shh expression, embryos were simultaneously hybridized with probes ofShh and pax-2 (previously pax[b]), the early expression domain of whichmarks the posterior midbrain (Krauss, S. et al. (1991) Development 113:1193-1206). By this stage, the anterior boundary of the Shh expressiondomain is positioned in the centre of the animal pole and coincidesapproximately with that of axial. At the same stage, prechordal platecells expressing the homeobox gene goosecoid (gsc) overlap and underlaythe presumptive forebrain (Statchel, S. E. et al., (1993) Development117: 1261-1274). Whereas axial is also thought to be expressed in headmesodermal tissue at this stage, we cannot be certain whether Shh isexpressed in the same cells. Sections of stained embryos suggest that inthe head Shh may by this stage be expressed exclusively inneuroectodermal tissue.

[0394] (v) Somitogenesis

[0395] By the onset of somitogenesis (approximately 10.5 h ofdevelopment), Shh expression in the head is clearly restricted to theventral floor of the brain, extending from the tip of the diencephaloncaudally through the hindbrain. At this stage, expression of axial hasalso disappeared from the head mesoderm and is similarly restricted tothe floor of the brain; in contrast to Shh, however, it extends only asfar as the anterior boundary of the midbrain. At this point, gscexpression has become very weak and is restricted to a ring of cellsthat appear to be migrating away from the dorsal midline.

[0396] As somitogenesis continues, Shh expression extends in arostral-caudal progression throughout the ventral region of the centralnervous system (CNS). Along the spinal cord, the expression domain isrestricted to a single row of cells, the floor plate, but graduallybroadens in the hindbrain and midbrain to become 5-7 cells in diameter,with a triangular shaped lateral extension in the ventral diencephalonand two strongly staining bulges at the tip of the forebrain, presumablyin a region fated to become hypothalamus.

[0397] As induction of Shh in the floor plate occurs, expression in theunderlying mesoderm begins to fade away, in a similar manner to axial(Strähle, U. et al., (1993) Genes & Dev. 7: 1436-1446). Thisdownregulation also proceeds in a rostral to caudal sequence, coincidingwith the changes in cell shape that accompany notochord differentiation.By the 22 somite stage, mesodermal Shh expression is restricted to thecaudal region of the notochord and in the expanding tail bud where abulge of undifferentiated cells continue to express Shh at relativelyhigh levels. Expression in the midbrain broadens to a rhombic shapedarea; cellular rearrangements that lead to the 90° kink of forebrainstructures, position hypothalamic tissue underneath the ventralmidbrain. These posterior hypothalamic tissues do not express Shh. Inaddition to Shh expression in the ventral midbrain, a narrow stripe ofexpressing cells extends dorsally on either side of the third ventriclefrom the rostral end of the Shh domain in the ventral midbrain to theanterior end of, but not including, the epiphysis. The most rostral Shhexpressing cells are confined to the hypothalamus. In the telencephalon,additional Shh expression is initiated in two 1-2 cell wide stripes.

[0398] By 36 hours of development, Shh expression in the ventral CNS hasundergone further changes. While expression persists in the floor plateof the tailbud, more rostrally located floor plate cells in the spinalcord cease to express the gene. In contrast, in the hindbrain andforebrain Shh expression persists and is further modified.

[0399] At 26-28 h, Shh expression appears in the pectoral fin primordia,that are visible as placode like broadenings of cells underneath theepithelial cell layer that covers the yolk. By 33 hrs of developmenthigh levels of transcript are present in the posterior margin of thepectoral buds; at the same time, expression is initiated in a narrowstripe at the posterior of the first gill. Expression continues in thepectoral fin buds in lateral cells in the early larva. At this stage,Shh transcripts are also detectable in cells adjacent to the lumen ofthe foregut.

[0400] (vi) Expression of Shh in Cyclops and Notail Mutants

[0401] Two mutations affecting the differentiation of the Axial tissuesthat express Shh have been described in zebrafish embryos homozygous forthe cyclops (cyc) mutation lack a differentiated floorplate (Hatta, K.et al., (1991) Nature 350: 339-341). By contrast, homozygous notail(ntl) embryos are characterized by a failure in notochord maturation anda disruption of normal development of tail structures (Halpern, M. E. etal., (1993) Cell 75: 99-111).

[0402] A change in Shh expression is apparent in cyc embryos as early asthe end of gastrulation; at this stage, the anterior limit of expressioncoincides precisely with the two pax-2 stripes in the posteriormidbrain. Thus, in contrast to wild-type embryos, no Shh expression isdetected in midline structures of the midbrain and forebrain. By the 5somite stage, Shh transcripts are present in the notochord which at thisstage extends until rhombomere 4; however, no expression is detected inmore anterior structures. Furthermore, no Shh expression is detected inthe ventral neural keel, in particular in the ventral portions of themidbrain and forebrain.

[0403] At 24 hours of development, the morphologically visible cycphenotype consists of a fusion of the eyes at the midline due to thecomplete absence of the ventral diencephalon. As at earlierdevelopmental stages, Shh expression is absent from neural tissue. Shhexpression in the extending tail bud of wild-type embryos is seen as asingle row of floor plate cells throughout the spinal cord. In a cycmutant, no such Shh induction occurs in cells of the ventral spinal cordwith the exception of some scattered cells that show transientexpression near the tail. Similarly, no Shh expression is seen rostrallyin the ventral neural tube. However, a small group of Shh expressingcells is detected underneath the epiphysis which presumably correspondto the dorsal-most group of Shh expressing cells in the diencephalon ofwild-type embryos.

[0404] In homozygous notail (ntl) embryos, no Shh staining is seen inmesodermal tissue at 24 hours of development, consistent with the lackof a notochord in these embryos; by contrast, expression throughout theventral CNS is unaffected. At the tail bud stage, however, just prior tothe onset of somitogenesis, Shh expression is clearly detectable innotochord precursor cells.

[0405] (vii) Injection of Synthetic Shh Transcripts Into ZebrafishEmbryos Induces Expression of a Floor Plate Marker

[0406] To investigate the activity of Shh in the developing embryo, anover-expression strategy, similar to that employed in the analysis ofgene function in Xenopus, was adopted. Newly fertilized zebrafish eggswere injected with synthetic Shh RNA and were fixed 14 or 24 hourslater. As an assay for possible changes in cell fate consequent upon theectopic activity of Shh, we decided to analyze Axial expression, sincethis gene serves as a marker for cells in which Shh is normallyexpressed. A dramatic, though highly localized ectopic expression ofAxial in a significant proportion (21/80) of the injected embryos fixedafter 24 hours of development is observed. Affected embryos show abroadening of the Axial expression domain in the diencephalon andectopic Axial expression in the midbrain; however, in no case hasectopic expression in the telencephalon or spinal cord been observed.Many of the injected embryos also showed disturbed forebrain structures,in particular smaller ventricles and poorly developed eyes. Amongstembryos fixed after 14 h, a similar proportion (8/42) exhibit the samebroadening and dorsal extension of the Axial stripe in the diencephalonas well as a dorsal extension of Axial staining in the midbrain; again,no changes in Axial expression were observed caudal to the hindbrainwith the exception of an increased number of expressing cells at the tipof the tail.

[0407] (viii) Overexpression of Shh in Drosophila Embryos Activates thehh-Dependent Pathway

[0408] In order to discover whether the high degree of structuralhomology between the Drosophila and zebrafish hh genes also extends tothe functional level, an overexpression system was used to test theactivity of Shh in flies. Expression of Drosophila hh driven by theHSP70 promoter results in the ectopic activation of both the normaltargets of hh activity; the wg transcriptional domain expands to fillbetween one third to one half of each parasegment whereas ptc isectopically activated in all cells except those expressing en (Ingham,P. W. (1993) Nature 366:560-562). To compare the activities of the flyand fish genes, flies transgenic for a HS Shh construct were generateddescribed above and subjected to the same heat shock regime as H Shhtransgenic flies. HS Shh embryos fixed immediately after the second oftwo 30 min heat shocks exhibit ubiquitous transcription of the Shh cDNA.Similarly treated embryos were fixed 30 or 90 min after the second heatshock and assayed for wg or ptc transcription. Both genes were found tobe ectopically activated in a similar manner to that seen in heatshocked H Shh embryos; thus, the zebrafish Shh gene can activate thesame pathway as the endogenous hh gene.

EXAMPLE 5 Cloning, Expression and Localization of Human Hedgehogs

[0409] (i) Experimental Procedures

[0410] Isolation of Human Hedgehog cDNA Clones.

[0411] Degenerate nucleotides used to clone chick Shh (Riddle et al.,(1993) Cell 75:1401-1416) were used to amplify by nested PCR humangenomic DNA. The nucleotide sequence of these oligos is as follows:vHH5O: 5′-GGAATTCCCAG(CA)GITG(CT)AA(AG)GA(AG)(CA)(AG)I(GCT)TIAA-3′; (SEQID NO:18) vHH3O: 5′-TCATCGATGGACCCA(GA)TC(GA)AAICCIGC(TC)TC-3′; (SEQ IDNO:19) vHH3I: 5′-GCTCTAGAGCTCIACIGCIA(GA)IC(GT)IGGIA-3′ (SEQ ID NO:20)

[0412] The expected 220 bp PCR product was subcloned into pGEM7zf(Promega) and sequenced using Sequenase v2.0 (U.S. Biochemicals). Oneclone showed high nucleotide similarity to mouse Ihh and mouse Shhsequence (Echelard et al., (1993) Cell 75:1417-1430) and it was used forscreening a human fetal lung 5′-stretch plus cDNA library (Clontech) inλ gt10 phage. The library was screened following the protocol suggestedby the company and two positive plaques were identified, purified,subdcloned into pBluescript SK+ (Stratagene) and sequenced, identifyingthem as the human homologues of Shh (SEQ ID NO:6) and Ihh (SEQ ID NO:7).

[0413] One clone contained the fill coding sequence of a human homologof Shh as well as 150 bp of 5′ and 36 bp of 3′ untranslated sequence.The other clone, which is the human homolog of Ihh, extends from 330 bp3′ of the coding sequence to a point close to the predicted boundarybetween the first and second exon. The identity of these clones wasdetermined by comparison to the murine and chick genes. The proteinencoded by human Shh has 92.4% overall identity to the mouse Shh,including 99% identity in the amino-terminal half. The carboxyl-terminalhalf is also highly conserved, although it contains short stretches of16 and 11 amino acids not present in the mouse Shh. The human Ihhprotein is 96.8% identical to the mouse Ihh. The two predicted humanproteins are also highly related, particularly in their amino-terminalhalves where they are 91.4% identical. They diverge significantly intheir carboxyl halves, where they show only 45.1% identity. The highlevel of similarity in the amino portion of all of these proteinsimplies that this region encodes domains essential to the activity ofthis class of signaling molecules.

[0414] Northern Blotting

[0415] Multiple Tissue Northern Blot (Clontech) prepared from poly A+RNAisolated from human adult tissues was hybridized with either fill length³²P-labeled human Shh clone or ³²P-labeled human Ihh clone following theprotocol suggested by the company.

[0416] Digoxigenin in situ Hybridization.

[0417] Sections: tissues from normal human second trimester gestationabortus specimens were washed in PBS and fixed overnight at 4° C.paraformaldehyde in PBS, equilibrated 24 hours at 4° C. in 50% sucrosein PBS and then placed in 50% sucrose in oct for one hour beforeembedding in oct. Cryostat sections (10-25 mm) were collected onsuperfrost plus slides (Fisher) and frozen at −80° C. until used.Following a postfixation in 4% paraformaldehyde the slides wereprocessed as in Riddle et al., (1993) Cell 75:1401-1416 with thefollowing alterations: proteinase K digestion was performed at roomtemperature from 1-15 minutes (depending on section thickness),prehybridization, hybridization and washes time was decreased to 1/10 oftime.

[0418] Whole-mounts: tissues from normal second trimester human abortusspecimens were washed in PBS, fixed overnight at 4° C. in 4%paraformaldehyde in PBS and then processed as in Riddle et al., (1993)Cell 75:1401-1416.

[0419] Isolation of an Shh P1 Clone.

[0420] The human Shh gene was isolated on a P1 clone from a P1 library(Pierce and Sternberg, 1992) by PCR (polymerase chain reaction)screening. Two oligonucleotide primers were derived from the human Shhsequence. The two olignucleotide primers used for PCR were: SHHF5′-ACCGAGGGCTGGGACGAAGATGGC-3′ (SEQ ID NO:43) SHR5′-CGCTCGGTCGTACGGCATGAACGAC-3′ (SEQ ID NO:44)

[0421] The PCR reaction was carried using standard conditions asdescribed previously (Thierfelder et al., 1994) except that theannealing temperature was 65° C. These primers amplified a 119 bpfragment from human and P1 clone DNA. The P1 clone was designated SHHP1.After the P1 clone was isolated these oligonucleotides were used assequencing primers. A 2.5 KbEcoRI fragment that encoded a CA repeat wassubdcloned from this P1 clone using methods described previously(Thierfelder et al. 1994). Oligonucleotide primers that amplified thisCA repeat sequence were fashioned from the flanking sequences: SHHCAF5′-ATGGGGATGTGTGTGGTCAAGTGTA-3′ (SEQ ID NO:45) SHHCAR5′-TTCACAGACTCTCAAAGTGTATTTT-3′ (SEQ ID NO:46)

[0422] Mapping the Human Ihh and Shh Genes.

[0423] The human Ihh gene was mapped to chromosome 2 using somatic cellhybrids from NIGMS mapping pannel 2 (GM10826B).

[0424] The Shh gene was mapped to chromosome 7 using somatic cellhybrids from NIGMS mapping panel 2 (GM10791 and GM10868).

[0425] Linkage between the limb deformity locus on chromosome 7 and theShh gene was demonstrated using standard procedures. Family LD has beendescribed previously (Tkukurov et al., (1994) Nature Genet. 6:282-286).A CA repeat bearing sequence near the Shh gene was amplified from theDNA of all members of Family LD by PCR using the SHHCAF and SHHCARprimers. Linkage between the CA repeat and the LD disease genesegregating in Family LD was estimated by the MLINK program (October1967). Penetrance was set at 100% and the allele frequencies weredetermined using unrelated spouses in the LD family.

[0426] Interspecific Backcross Mapping

[0427] Interspecific backcross progeny were generated by mating(C57BL/6J×M. spretus) F1 females and C57BL/6J males as described(Copeland and Jenkins, (1991) Trends Genet. 7:113-118). A total of 205N2 mice were used to map the Ihh and Dhh loci. DNA isolation,restriction enzyme digestions, agarose gel electrophoresis, Southernblot transfer and hybridization were performed essentially as described(Jenkins et al., (1982) J. Virol. 43:26-36). All blots were preparedwith Hybond-N+ nylon membrane (Amersham). The probe, an ˜1.8 kb EcoRIfragment of mouse cDNA, detected a major fragment of 8.5 kb in C57BL/6j(B) DNA and a major fragment 6.0 kb in M. spretus (S) DNA followingdigestion with Bgl II. The Shh probe, an ˜900 bp SmaI fragment of mousecDNA, detected HincII fragments of 7.5 and 2.1 kb (B) as well as 4.6 and2.1 (S). The Dhh probe, and ˜800 bp BamHi/EcoRi fragment of mousegenomic DNA, detected major fragments of 4.7 and 1.3 kb (B) and 8.2 and1.3 kb (S) following digestion with SphI. The presence or absence of M.spretus specific fragments was followed in backcross mice.

[0428] A description of the probes and RFLPs for loci used to positionthe Ihh, Shh and Dhh loci in the interspecific backcross has beenreported. These include: Fn1, Vil and Acrg, chromosome 1 (Wilkie et al.,(1993) Genomics 18:175-184), Gnai1, En2, I16, chromosomes 5 (Miao etal., (1994) PNAS USA 91:11050-11054) and Pdgfb, Gdc1 and Rarg,chromosome 15 (Brannan et al., (1992) Genomics 13:1075-1081).Recombination distances were calculated as described (Green, (1981)Linkage, recombination and mapping. In “Genetics and Probability inAnimal Breeding Experiments”, pp. 77-113, Oxford University Press, NY)using the computer program SPRETUS MADNESS. Gene order was determined byminimizing the number of recombination events required to explain theallele distribution patterns.

[0429] (ii) Expression of Human Shh and Thh

[0430] To investigate the tissue distribution of Shh and Ihh expression,poly(A)+RNA samples from various adult human tissues were probed withthe two cDNA clones. Of the tissues tested, an Ihh-specific message of2.7˜kb is only detected in liver and kidney. Shh transcripts was notdetected in the RNA from any of the adult tissues tested. All thesamples contained approximately equal amounts of intact RNA, asdetermined by hybridization with a control probe.

[0431] The hedgehog family of genes were identified as mediators ofembryonic patterning in flies and vertebrates. No adult expression ofthese genes had previously been reported. These results indicate thatIhh additionally plays a role in adult liver and kidney. Since thehedgehog genes encode intercellular signals, Ihh may function incoordinating the properties of different cell types in these organs. Shhmay also be used as a signaling molecule in the adult, either in tissuesnot looked at here, or at levels too low to be detected under theseconditions.

[0432] In situ hybridization was used to investigate the expression ofShh in various mid-gestational human fetal organs. Shh expression ispresent predominantly in endoderm derived tissues: the respiratoryepithelium, collecting ducts of the kidney, transitional epithelium ofthe ureter, hepatocytes, and small intestine epithelium. Shh was notdetectable in fetal heart or placental tissues. The intensity ofexpression is increased in primitive differentiating tissues (renalblastema, base villi, branching lung buds) and decreased or absent indifferentiated tissues (e.g. glomeruli). Shh expression is present inthe mesenchyme immediately abutting the budding respiratory tubes. Thenon-uniform pattern of Shh expression in hepatocytes is consistent withexpression of other genes in adult liver (Dingemanse et al., (1994)Differentiation 56:153-162). The base of villi, the renal blastema, andthe lung buds are all regions expressing Shh and they are areas ofactive growth and differentiation, suggesting Shh is important in theseprocesses.

[0433] (iii) The Chromosomal Map Location of Human Shh and Ihh.

[0434] Since Shh is known to mediate patterning during the developmentof the mouse and chick and the expression of Shh and Ihh are suggestiveof a similar role in humans, mutations in these genes would be expectedto lead to embryonic lethality or congenital defects. One way ofinvestigating this possibility is to see whether they are geneticallylinked to any known inherited disorders.

[0435] Shh- and Ihh-specific primers were designed from their respectivesequences and were used in PCR reactions on a panel of rodent-humansomatic cell hybrids. Control rodent DNA did not amplify specific bandsusing these primers. In contrast, DNA from several rodent-human hybridsresulted in PCR products of the appropriate size allowing us to assignShh to chromosome 7q and Ihh to chromosome 2.

[0436] One of the central roles of chick Shh is in regulating theanterior-posterior axis of the limb. A human congenital polysyndactylyhas recently been mapped to chromosome 7q36 (Tsukurov et al., (1994)Nature Genet. 6:282-286; Heutink et al., (1994) Nature Genet.6:287-291). The phenotype of this disease is consistent with defectsthat might be expected from aberrant expression of Shh in the limb.Therefore, the chromosomal location of Shh was mapped more precisely, inparticular in relation to the polysyndactyly locus.

[0437] A P1 phage library was screened using the Shh specific primersfor PCR amplification and clone SHHP1 was isolated. Clone SHHP1contained Shh sequence. A Southern blot of an EcoRi digest of this phageusing [CA]/[GT] probe demonstrated that a 2.5 Kb EcoRi fragmentcontained a CA repeat. Nucleotide sequence analysis of this subdclonedEcoRI fragment demonstrated that the CA repeat lay near the EcoRI sites.Primers flanking the CA repeat were designed and used to map thelocation of Shh relative to other markers on 7q in individuals of alarge kindred with complex polysyndactyly (Tsukurov et al., (1994)Nature Genet. 6:282-286). Shh maps close to D75550 on 7q36, with norecombination events seen in this study. It is also extremely close to,but distinct from, the polysyndactyly locus with one recombination eventobserved between them (maximum lod score=4.82, Θ=0.05). One unaffectedindividual (pedigree ID V-10 in Tsukurov et al., (1994) Nature Genet.6:282-286) has the Shh linked CA repeat allele found in all affectedfamily members. No recombination was observed between the locus En2 andthe Shh gene (maximum lod score=1.82, Θ=0.0).

[0438] (iv) Chromosomal Mapping of the Murine Ihh, Shh and Dhh Genes.

[0439] The murine chromosomal location of Ihh, Shh and Dhh wasdetermined using an interspecific backcross mapping panel derived fromcrosses of [(C57BL/6J×M. spetrus)F1 X C57BL/J)] mice. cDNA fragmentsfrom each locus were used as probes in Southern blot hybridizationanalysis of C57BL/6J and M. spretus genomic DNA that was separatelydigested with several different restriction enzymes to identifyinformative restriction fragment length polymorphisms (RFLPs) useful forgene mapping. The strain distribution pattern of each RFLP in theinterspecific backcross was then determined by following the presence orabsence of RFLPs specific for M. spretus in backcross mice.

[0440] Ihh mapped to the central region of mouse chromosome 1, 2.7 cMdistal of Fn1 and did not recombine with Vil in 190 animals typed incommon, suggesting that the two loci are within 1.6 cM (upper 95%confidence level) (FIG. 16). Shh mapped to the proximal region of mousechromosome 5, 0.6 cM distal of En2 and 1.9 cM proximal of I16 inaccordance to Chang et al., (1994) Development 120:3339-3353. Dhh mappedto the very distal region of mouse chromosome 15, 0.6 cM distal of Gdc1and did not recombine with Rarg in 160 animals typed in common,suggesting that the two loci are within 1.9 cM of each other (upper 95%confidence level) (FIG. 16).

[0441] Interspecific maps of chromosome 1, 5 and 15 were compared withcomposite mouse linkage maps that report the map location of manyuncloned mouse mutations (compiled by M. T. Davisson, T. H. Roderick, A.L. Hillyard and D. P. Doolittle and provided from GBASE, a computerizeddatabase maintained at The Jackson Laboratory, Bar Harbor, Me.). Thehemimelic extra-toe (Hx) mouse mutant maps 1.1 cM distal to En2 onchromosome 5 (Martin et al., (1990) Genomics 6:302-308), a location inclose proximity to where Shh has been positioned. Hx is a dominantmutation which results in preaxial polydactyly and hemimelia affectingall four limbs (Dickie, (1968) Mouse News Lett 38:24; Knudsen andKochhar, (1981) J. Embryol. Exp. Morph. 65: Suppl. 289-307). Shh haspreviously been shown to be expressed in the limb (Echelard et al.,(1993) Cell 75:1417-1430). To determine whether Shh and Hx are tightlylinked we followed their distribution in a backcross panel in which Hxwas segregating. Two recombinants between Shh and Hx were identified,thus excluding the possibility that the two loci are allelic and theseobservations are again consistent with those of Chang et al., (1994)Development 120:3339-3353. Wile there are several other mutations in thevicinity of Ihh and Dhh, none is an obvious candidate for an alterationin the corresponding gene.

[0442] The central region of mouse chromosome 1 shares homology withhuman chromosome 2q (summarized in FIG. 16). Placement of Ihh in thisinterval suggests the human homolog of Ihh will reside on 2q, as well.Similarly, it is likely that human homolog of Dhh will reside on humanchromosome 12q.

EXAMPLE 6 Proteolytic Processing Yields Two Secreted Forms of SonicHedgehog

[0443] (i) Experimental Procedures

[0444] In vitro Translation and Processing

[0445] Mouse and chick sonic hedgehog coding sequences were insertedinto the vector pSP64T (kindly provided by D. Melton) which contains anSP6 phage promoter and both 5′ and 3′ untranslated sequences derivedfrom the Xenopus laevis β-Globin gene. After restriction endonucleasedigestion with Sal I to generate linear templates, RNA was transcribedin vitro using SP6 RNA polymerase (Promega, Inc.) in the presence of 1mM cap structure analog (m⁷G(5′)ppp(5′)Gm; Boehringer-Mannheim, Inc.)Following digestion with RQ1 DNase I (Promega, Inc.) to remove the DNAtemplate, transcripts were purified by phenol:choloroform extraction andethanol precipitation.

[0446] Rabbit reticulocyte lysate (Promega, Inc.) was used according tothe manufacturer's instructions. For each reaction, 12.5 μl of lysatewas programmed with 0.5-2.0 μg of in vitro transcribed RNA. Thereactions contained 20 μCi of Express labeling mix (NEN/DuPont, Inc.)were included. To address processing and secretion in vitro, 1.0-2.0 μlof canine pancreatic microsomal membranes (Promega, Inc.) were includedin the reactions. The final reaction volume of 25 μl was incubated forone hour at 30° C. Aliquots of each reaction (between 0.25 and 3.0 μl)were boiled for 3 minutes in Laemmli sample buffer (LSB: 125 mM Tris-Hcl[pH 6.8]; 2% SDS; 1% 2-mercaptoethanol; 0.25 mg/ml bromophenol blue)before separating on a 15% polyacrylamide gel. Fixed gels were processedfor fluorography using EnHance (NEN/DuPont, Inc.) as described by themanufacturer.

[0447] Glycosylation was addressed by incubation with Endoglycosidase H(Endo H; New England Biolabs, Inc.) according to the manufacturer'sdirections. Reactions were carried out for 1-2 hr at 37° C. beforeanalyzing reaction products by polyacrylamide gel electrophoresis(PAGE).

[0448] Xenopus Oocyte Injection and Labeling

[0449] Oocytes were enzymatically defolliculated and rinsed with OR2 (50mM HEPES [pH 7.2], 82 mM NaCl, 2.5 mM KCl, 1.5 mM Na2HPO4). Healthystage six oocytes were injected with 30 ng of in vitro transcribed,capped mouse Shh RNA (prepared as described above). Following a 2 hrrecovery period, healthy injected oocytes and uninjected controls werecultured at room temperature in groups of ten in 96-well dishescontaining 0.2 ml of OR2 (supplemented with 0.1 mg/ml Gentamicin and 0.4mg/ml BSA) per well. The incubation medium was supplemented with 50 μCiof Express labeling mix. Three days after injection, the culture mediawere collected and expression of Shh protein analyzed byimmunoprecipitation. Oocytes were rinsed several times in OR2 beforelysing in TENT (20 mM Tris-HCl [pH 8.0]; 150 mM NaCl, 2 mM EDTA; 1%Triton-X-100; 10 μl/oocyte) supplemented with 1 μg/ml aprotinin, 2 μg/mlleupeptin and 1 mM phenylmethylsufonylfluoride (PMSF). Aftercentrifugation at 13000×g for 10 minutes at 4° C., soluble proteinsupernatants were recovered and analyzed by immunoprecipitation (seebelow).

[0450] Cos Cell Transfection and Labeling

[0451] Cos cells were cultured in Dulbecco's Modified Eagle Medium(DMEM; Sigma, Inc.) supplemented with 10% fetal bovine serum(Gibco/BRL), 2 mM L-Glutamine (Gibco/BRL) and 50 mU/ml penicillin and 50μg/ml streptomycin (Gibco/BRL). Subconfluent cos cells in 35 mm or 60 mmdishes (Falcon, Inc.) were transiently transfected with 2 mg or 6 mgsupercoiled plasmid DNA, respectively. Between 42 and 44 hrpost-transfection, cells were labeled for 4-6 hr in 0.5 ml (35 mmdishes) or 1.5 ml (60 mm dishes) serum-free DMEM lacking Cysteine andMethionine (Gibco/BRL) and supplemented with 125 μCi/ml each of Expresslabeling mix and L-35S-Cysteine (NEN/DuPont). After labeling, media werecollected and used for immunoprecipitation. Cells were rinsed with coldPBS and lysed in the tissue culture dishes by the addition of 0.5 ml (35mm dishes) or 1.5 ml (60 mm dishes) TENT (with protease inhibitors asdescribed above) and gentle rocking for 30 minutes at 4° C. Lysates werecleared by centrifugation (13000×g for 5 min. at 4° C.) and thesupernatants were analyzed by immunoprecipitation (see below).

[0452] Baculovirus Production and Infection

[0453] A recombinant baculovirus expressing mouse sonic hedgehog with amyc epitope tag inserted at the carboxy terminus was generated using theBaculogold kit (Pharmingen, Inc.). The initial virus production used Sf9 cells, followed by two rounds of amplification in High Five cells(Invitrogen, Inc.) in serum-free medium (ExCell 401; Invitrogen, Inc.).A baculovirus lacking Shh coding sequences was also constructed as acontrol. For protein induction, High Five cells were infected at amultiplicity of approximately 15. Three days later, medium and cellswere collected by gentle pipetting. Cells were collected bycentrifugation (1000×g) and the medium was recovered for Western blotanalysis. Cell pellets were washed twice in cold PBS and lysed in TENTplus protease inhibitors (see above) by rotating for 30 minutes at 4° C.in a microcentrifuge tube. The lysate was cleared as described aboveprior to Western blotting.

[0454] Western Blotting

[0455] For Western blotting, 0.25 ml samples of media (1% of the total)were precipitated with TCA and redissolved in 15 μl of LSB. Cell lysatesamples (1% of total) were brought to a final volume of 15 μl with waterand concentrated (5×) LSB . Samples were boiled S minutes prior toseparation on a 15% acrylamide gel. Proteins were transferred to PVDFmembrane (Immobilon-P; Millipore, Inc.) and blocked in BLOTTO (5% w/vnon-fat dried milk in PBS) containing 0.2% Tween-20. Hybridomasupernatant recognizing the human c-myc epitope (9E10;, Evan, G. I. etal., (1985) Mol. Cell. Biol. 5:3610-3616) was added at a dilution of1:200 for one hour followed by a 1:5000 dilution of Goatanti-Mouse-Alkaline phosphatase conjugate (Promega, Inc.) for 30minutes. Bands were visualized using the Lumi-Phos 530 reagent(Boehringer-Mannheim) according to the manufacturer's directions.

[0456] For Western blotting of COS cell material, cleared media (seeabove) were precipitated with TCA in the presence of 4 μg of BSA per mlas a carrier. the protein pellets were dissolved in 20 μl of LSB.Dissolved medium protein and cell lysates (see above) were boiled for 5min, and 10 μl (50%) of each medium sample and 10 μl (10%) of each celllysate were separated on a 15% acrylamide gel. The gel was blotted to apolyvinylidene difluoride membrane as described above. The membrane wasblocked as described above and incubated in a 1:200 dilution ofaffinity-purified Shh antiserum (see below) and then in a 1:5,000dilution of horseradish peroxidase-conjugated donkey anti-rabbitimmunoglobulin g (IgG; Jackson Immuno research, Inc.). Bands werevisualized with the Enhanced Chemiluminescence kit (Amersham, Inc.)according to the manufacturer's instructions.

[0457] For Western blotting of mouse and chicken embryonic tissuelysates, 60 μg of each sample was separated on 15% acrylamide gels.Blotting and probing with affinity-purified Shh antiserum as well aschemiluminescence detection were carried out as described above for theCOS cell material.

[0458] Immunoprecipitation

[0459] Cell lysates (Xenopus oocytes or cos cells) were brought to 0.5ml with TENT (plus protease inhibitors as above). Media samples (OR2 orDMEM) were cleared by centrifugation at 13000×g for 5 min. (4° C.) and10× TENT was added to a final concentration of 1× (final volume: 0.5-1.5ml). The c-myc monoclonal antibody hybridoma supernatant was added to1/20 of the final volume. Samples were rotated for 1 hr at 4° C., then0.1 ml of 10% (v/v) protein A-Sepharose CL-4B (Pharmacia, Inc.) wasadded. Samples were rotated an additional 14-16 h. Immune complexes werewashed 4 times with 1.0 ml TENT. Immunoprecipitated material was elutedand denatured by boiling for 10 minutes in 25 μl 1× LSB. Followingcentrifugation, samples were separated on 15% acrylamide gels andprocessed for fluorography as described previously. Samples for Endo Hdigestion were eluted and denatured by boiling for 10 minutes in theprovided denaturation buffer followed by digestion with Endo H for 1-2hr at 37° C. Concentrated (SX) LSB was added and the samples wereprocessed for electrophoresis as described.

[0460] For immunoprecipitation with the anti-mouse Shh serum, samples(Cos cell lysates and DMEM) were precleared by incubating 1 hr on icewith 3 μl pre-immune serum, followed by the addition of 0.1 ml 10% (v/v)Protein A-Sepharose. After rotating for 1 hr at 4 C, supernatants wererecovered and incubated for 1 hr on ice with 3 μl depleted anti-mouseShh serum (see below). Incubation with Protein A-Sepharose, washing,elution and electrophoresis were then performed as described above.

[0461] Immunofluorescent Staining of Cos Cells

[0462] Twenty-four hours after transfection, cells were transferred to8-chamber slides (Lab-Tek, Inc.) and allowed to attach an additionaltwenty-four hours. Cells were fixed in 2% paraformaldehyde/0.1%glutaraldehyde, washed in PBS and permeabilized in 1% Triton-X-100(Munro, S. and Pelham, H. R. B., (1987) Cell 48:899-907). After washingin PBS, cells were treated for 10 minutes in 1 mg/ml sodium borohydride.Cells were incubated with the c-myc monoclonal antibody hybridomasupernatant (diluted 1:10) and the affinity purified mouse Sonichedgehog antiserum (diluted 1:4) for 45 minutes followed by incubationin 1:100 Goat-anti Mouse IgG-RITC plus 1:100 Goat anti Rabbit IgG F1 TC(Southern Biotechnology Associates, Inc.) for 45 minutes. DAPI (Sigma,Inc.) was included at 0.3 μg/ml The slides were mounted in Slo-Fade(Molecular Probes, Inc.) and photographed on a Leitz DMR compoundmicroscope.

[0463] Embryonic Tissue Dissection and Lysis

[0464] Mouse forebrain, midbrain, hindbrain, lung, limb, stomach, andliver tissues form 15-15.5-day-postcoitum Swiss Webster embryos weredissected into cold PBS, washed several times in PBS, and then lysed bytrituraton and gentle sonication in LSB lacking bromophenol blue.Lysates were cleared by brief centrifugation, and protein concentrationswere determined by the Bradford dye-binding assay.

[0465] To obtain chicken CNS and limb bud tissue, fertilized eggs(Spafas, Inc.) were incubated at 37° C. until the embryos reached stages20 and 25, respectively (Hamburg and Hamilton (1951) J. Exp. Morphol.88:49-92). By using sharp tungsten needles, dorsal and ventral pieces ofthe anterior CNS were obtained from the stage 15 embryos, and limb budsfrom the stage 25 embryos were cut into anterior and posterior halves.Tissues were lysed, and protein concentrations were determined asdescribed above. Prior to electrophoresis of the mouse and chickenproteins (see above), samples were brought to 20 μl with LSB containingbromophenol blue and boiled for 5 minutes.

[0466] Antibody Production and Purification

[0467] A PCR fragment encoding amino acids 44-143 of mouse Sonichedgehog was cloned in frame into the Eco Rl site of pGEX-2T (Pharmacia,Inc.). Transformed bacteria were induced with IPTG and the fusionprotein purified on a Glutathione-Agarose affinity column (Pharmacia,Inc.) according to the manufacturers instructions. Inoculation of NewZealand White rabbits, as well as test and production bleeding werecarried out at Hazelton Research Products, Inc.

[0468] To deplete the serum of antibodies againstGlutathione-S-transferase (GST) and bacterial proteins, a lysate of E.coli transformed with pGEX-2T and induced with IPTG was coupled toAffi-Gel 10 (Bio-Rad, Inc.) The serum was incubated in batch for twohours with the depletion matrix before centrifugation (1000×g for 5 mm.)and collection of the supernatant. To make an affinity matrix, purifiedbacterially expressed protein corresponding to the amino terminaltwo-thirds of mouse Sonic hedgehog was coupled to Affi-Gel 10 (Bio-Rad,Inc.). The depleted antiserum was first adsorbed to this matrix inbatch, then transferred to a column. The matrix was washed with TBST (25mM Tris-HCl [pH 7.5], 140 mM NaCl, 5 mM KCl, 0.1% Triton-X-100), and thepurified antibodies were eluted with ten bed volumes of 0.15 M Glycine[pH 2.5]. The solution was neutralized with one volume of 1 M Tris-HCl[pH 8.0], and dialyzed against 160 volumes of PBS.

[0469] Other antibodies have been generated against hedgehog proteinsand three polyclonal rabbit antisera obtained to hh proteins can becharacterized as follows:Ab77-reacts only with the carboxyl processedchick Shh peptide (27 kd); Ab79 -reacts with amino processed chick,mouse and human Shh peptide (19 kd). Weakly reacts with 27 kd peptidefrom chick and mouse. Also reacts with mouse Ihh; and Ab80-reacts withonly amino peptide (19 kd) of chick, mouse and human.

[0470] (ii) In Vitro Translated Sonic Hedgehog is ProteolyticallyProcessed and Glycosylated

[0471] The open reading frames of chick and mouse Shh encode primarytranslation products of 425 and 437 amino acids, respectively, withpredicted molecular masses of 46.4 kilodaltons (kDa) and 47.8 kDa(Echelard, Y. et al., (1993) Cell 75:1417-1430; Riddle, R. D. et al.,(1993) Cell 75:1401-1416). Further examination of the protein sequencesrevealed a short stretch of amino terminal residues (26 for chick, 24for mouse) that are highly hydrophobic and are predicted to encodesignal peptides. Removal of these sequences would generate proteins of43.7 kDa (chick Shh) and 45.3 kDa (mouse Shh). Also, each proteincontains a single consensus site for N-linked glycosylation (Tarentino,A. L. et al., (1989) Methods Cell Biol. 32:111-139) at residue 282(chick) and 279 (mouse). These features of the Shh proteins aresummarized in FIG. 11.

[0472] A rabbit reticulocyte lysate programmed with in vitro translatedmessenger RNA encoding either chick or mouse Shh synthesizes proteinswith molecular masses of 46 kDa and 47 kDa, respectively. These valuesare in good agreement with those predicted by examination of the aminoacid sequences. To examine posttranslational modifications of Shhproteins, a preparation of canine pancreatic microsomal membranes wasincluded in the translation reactions. This preparation allows suchprocesses as signal peptide cleavage and core glycosylation. When theShh proteins are synthesized in the presence of these membranes, twoproducts with apparent molecular masses of approximately 19 and 28 kDa(chick), or 19 and 30 kDa (mouse) are seen in addition to the 46 kDa and47 kDa forms. When the material synthesized in the presence of themembranes is digested with Endoglycosidase H (Endo H), the mobilities ofthe two larger proteins are increased. The apparent molecular masses ofthe Endo H digested forms are 44 kDa and 26 kDa for chick Shh, and 45kDa and 27 kDa for mouse Shh. The decrease in the molecular masses ofthe largest proteins synthesized in the presence of the microsomalmembranes after Endo H digestion is consistent with removal of thepredicted signal peptides. The mobility shift following Endo H treatmentindicates that N-linked glycosylation occurs, and that the 26 kDa(chick) and 27 kDa (mouse) proteins contain the glycosylation sites.

[0473] The appearance of the two lower molecular weight bands (hereafterreferred to as the “processed forms”) upon translation in the presenceof microsomal membranes suggests that a proteolytic event in addition tosignal peptide cleavage takes place. The combined molecular masses ofthe processed forms (19 kDa and 26 kDa for chick; 19 kDa and 27 kDa formouse) add up to approximately the predicted masses of the signalpeptide cleaved proteins (44 kDa for chick and 45 kDa for mouse)suggesting that only a single additional cleavage occurs.

[0474] The mouse Shh protein sequence is 12 amino acid residues longerthan the chick sequence (437 versus 425 residues). Alignment of thechick and mouse Shh protein sequences reveals that these additionalamino acids are near the carboxy terminus of the protein (Echelard, Y.et al., (1993) Cell 75:1417-1430). Since the larger of the processedforms differ in molecular mass by approximately 1 kDa between the twospecies, it appears that these peptides contain the carboxy terminalportions of the Shh proteins. The smaller processed forms, whosemolecular masses are identical, presumably consist of the amino terminalportions.

[0475] (iii) Secretion of Shh Peptides

[0476] To investigate the synthesis of Shh proteins in vivo, the mouseprotein was expressed in several different eukaryotic cell types. Inorder to detect synthesized protein, and to facilitate futurepurification, the carboxy terminus was engineered to contain atwenty-five amino acid sequence containing a recognition site for thethrombin restriction protease followed by a ten amino acid sequencederived from the human c-myc protein and six consecutive histidineresidues. The c-myc sequence serves as an epitope tag allowing detectionby a monoclonal antibody (9E10; Evan, G. I. et al., (1985) Mol. CellBiol. 5:3610-3616). The combined molecular mass of the carboxy terminaladditions is approximately 3 kDa.

[0477]Xenopus laevis oocytes

[0478] Immunoprecipitation with the c-myc antibody detects severalproteins in lysates of metabolically labeled Xenopus laevis oocytesinjected with Shh mRNA. Cell lysates and medium from ³⁵S labeled oocytesinjected with RNA encoding mouse Shh with the c-myc epitope tag at theat the carboxy terminus, or from control oocytes were analyzed byimmunoprecipitation with c-myc monoclonal antibody. A band ofapproximately 47 kDa is seen, as is a doublet migrating near 30 kDa.Treatment with Endo H increases the mobility of the largest protein, andresolves the doublet into a single species of approximately 30 kDa.These observations parallel the behaviors seen in vitro. Allowing forthe added mass of the carboxy terminal additions, the largest proteinwould correspond to the signal peptide cleaved form, while the doubletwould represent the glycosylated and unglycosylated larger processedform. Since the epitope tag was placed at the carboxy terminus of theprotein, the identity of the 30 kDa peptide as the carboxy terminalportion of Shh is confirmed. Failure to detect the 19 kDa speciessupports its identity as an amino terminal region of the protein.

[0479] To test whether Shh is secreted by Xenopus oocytes, the medium inwhich the injected oocytes were incubated was probed byimmunoprecipitation with the c-myc antibody. A single band migratingslightly more slowly than the glycosylated larger processed form wasobserved. This protein is insensitive to Endo H. This result is expectedsince most secreted glycoproteins lose sensitivity to Endo H as theytravel through the Golgi apparatus and are modified by a series ofglycosidases (Kornfeld, R. and Kornfeld, S., (1985) Annu. Rev. Biochem.54:631-664). The enzymatic maturation of the Asn-linked carbohydratemoiety could also explain the slight decrease in mobility of thesecreted larger protein versus the intracellular material. FollowingEndo H digestion, a band with a slightly lower mobility than the signalpeptide cleaved protein is also apparent, suggesting that some Shhprotein is secreted without undergoing proteolytic processing. Failureto detect this protein in the medium without Endo H digestion suggestsheterogeneity in the extent of carbohydrate modification in the Golgipreventing the material from migrating as a distinct band. Resolution ofthis material into a single band following Endo H digestion suggeststhat the carbohydrate structure does not mature completely in the Golgiapparatus. Structural differences between the unprocessed protein andthe larger processed form could account for this observation (Kornfeld,R. and Kornfeld, S., (1985) Annu. Rev. Biochem. 54:631-664).

[0480] Cos Cells

[0481] The behavior of mouse Shh in a mammalian cell type wasinvestigated using transfected cos cells. Synthesis and secretion of theprotein was monitored by immunoprecipitation using the c-myc antibody.Transfected cos cells express the same Sonic hedgehog species that weredetected in the injected Xenopus oocytes, and their behavior followingEndo H digestion is also identical. Furthermore, secretion of the 30 kDaglycosylated form is observed in cos cells, as well as thecharacteristic insensitivity to Endo H after secretion. Most of thesecreted protein co-migrates with the intracellular, glycosylated largerprocessed form, but a small amount of protein with a slightly lowermobility is also detected in the medium. As in the Xenopus oocytecultures, some Shh which has not undergone proteolytic processing isevident in the medium, but only after Endo H digestion.

[0482] Baculovirus Infected Cells

[0483] To examine the behavior of the mouse Shh protein in aninvertebrate cell type, and to potentially purify Shh peptides, arecombinant baculovirus was constructed which placed the Shh codingsequence, with the carboxy terminal tag, under the control of thebaculoviral Polyhedrin gene promoter. When insect cells were infectedwith the recombinant baculovirus, Shh peptides could be detected in celllysates and medium by Western blotting with the c-myc antibody.

[0484] The Shh products detected in this system were similar to thosedescribed above. However, virtually no unprocessed protein was seen incell lysates, nor was any detected in the medium after Endo H digestion.This suggests that the proteolytic processing event occurs moreefficiently in these cells than in either of the other two cell types orthe in vitro translation system. A doublet corresponding to theglycosylated and unglycosylated 30 kDa forms is detected, as well as thesecreted, Endo I resistant peptide as seen in the other expressionsystems. Unlike the other systems, however, all of the secreted largerprocessed form appears to comigrate with the glycosylated intracellularmaterial.

[0485] (iv) Secretion of a Highly Conserved Amino Terminal Peptide

[0486] To determine the behavior of the amino terminal portion of theprocessed Sonic hedgehog protein, the c-myc epitope tag was positioned32 amino acids after the putative signal peptide cleavage site (FIG.12). Cos cells were transfected with Shh expression constructscontaining the c-myc tag at the carboxy terminus or near the aminoterminus When this construct was expressed in cos cells, both the fulllength protein and the smaller processed form (approximately 20 kDa dueto addition of the c-myc tag) were detected by immunoprecipitation ofextracts from labeled cells. However, the 20 kDa product is barelydetected in the medium. In cells transfected in parallel with thecarboxy terminal c-myc tagged construct, the full length and 30 kDaproducts were both precipitated from cell lysates and medium asdescribed earlier.

[0487] As the amino terminal c-myc tag may affect the secretionefficiency of the smaller processed form, the expression of this proteinwas examined in cos cells using an antiserum directed against aminoacids 44 through 143 of mouse Shh (FIG. 12). After transfection with thecarboxy-terminal c-myc tagged construct, immunoprecipitation with theanti-Shh serum detected a very low level of the smaller processed formin the medium despite a strong signal in the cell lysate. Thisrecapitulates the results with the myc antibody.

[0488] To examine the subcellular localization of Shh proteins, coscells were transfected with the carboxy terminal tagged Shh constructand plated on multi-chamber slides, fixed and permeabilized. The cellswere incubated simultaneously with the anti-Shh serum and the c-mycantibody followed by FITC conjugated Goat anti-Rabbit-IgG and RITCconjugated Goat anti-Mouse-IgG. DAPI was included to stain nuclei.Strong perinuclear staining characteristic of the Golgi apparatus wasobserved with the anti-Shh serum. The same subcellular region was alsostained using the c-myc antibody. The coincidence of staining patternsseen with the two antibody preparations suggest that the low level ofthe smaller processed form detected in the medium is not due to itsretention in the endoplasmic reticulum, since both processed formstraffic efficiently to the Golgi apparatus.

[0489] One explanation for the failure to detect large amounts of thesmaller processed form in the culture medium could that this proteinassociates tightly with the cell surface or ECM. To examine this, cellswere treated with the polyanionic compounds herparin and suramin. Thesecompounds have been shown to increase the levels of some secretedproteins in culture medium, possibly by displacing them from cellsurface or ECM components or by directly binding the proteins andperhaps protecting them from proteolytic degradation (Bradley and Brown(1990) EMBO J. 9:1569-1575; Middaugh et al. (1992) Biochem.31:9016-9024; Smolich et al. (1993) Mol. Biol. Cell 4:1267-1275). The19-kDa amino-terminal form of Shh is barely detectable in the medium oftransfected COS cells, despite its obvious presence in the cell lysate.However, in the presence of 10 mg of heparin per ml, this peptide isreadily detected in the medium. The addition of 10 mM suramin to themedium has an even greater effect. Since the concentrations used wherethose previously determined to elicit maximal responses, it is clearthat suramin is more active than heparin in this assay.

[0490] The ability of heparin and suramin to increase the amount of thesmaller processed form in the medium of transfected cells implies thatthis peptide may be tightly associated with the cell surface of ECM. Asa first step toward determining which region(s) of the Shh protein maybe responsible for this retention, a truncated form of mouse Shh deletedof all sequence downstream of amino acid 193 was expressed in COS cells.This protein contains all of the sequences encode by exons one and two,as well as five amino acids derived for exon three. Since its predictedmolecular mass (19.2 kDa) is very close to the observed molecular massof the smaller processed form, the behavior of this protein would beexpected to mimic that of the smaller processed form. This protein isdetected at a very high level in the medium, even in the absence ofheparin or suramin, and migrates at a position indistinguishable formthat of the amino-terminal cleavage product generated from thefull-length protein. In fact, virtually no protein is seen in the celllysates, suggesting nearly quantitative release of the protein into themedium. This raises the possibility that the actual amino terminallyprocessed form may extend a short distance beyond amino acid 193 andthat these additional amino acids contain a cell surface-ECM retentionsignal.

[0491] The influence of sequences located at the extreme amino andcarboxy termini of mouse Shh on the behavior of the protein intransfected cells was examined using the amino terminus-specificantiserum. Expression of a mouse Shh construct lacking a signal peptideresults in the accumulation of approximately 28-kDa protein, as well asa small amount of protein which comigrates with the smaller processedform. This implies that correct cleavage of Shh requires targeting ofthe protein to the endoplasmic reticulum, since the bulk of theprocessed form of Shh expressed in the cytoplasm is cleaved at a newposition that is approximately 9 kDa carboxy terminal to the normalcleavage site. Expression of a mouse Shh protein engineered to terminateafter amino acid 428 (lacking nine carboxy-terminal amino acids [ΔCt])results in the expected amino-terminal cleavage product; however, theefficiency of cleavage is significantly decreased compared with thatseen with the wild-type protein. Therefore, sequences located at adistance from the proteolytic processing site are able to affect theefficiency of processing.

[0492] (v) Sonic Hedgehog Processing in Embryonic Tissues

[0493] In order to determine whether the proteolytic processing of Shhobserved in the different expression systems reflects the behavior ofthe protein in embryos, the amino terminus-specific mouse Shh antiserumwas used to probe Western blots of various chicken and mouse embryonictissues. A protein with an electrophoretic mobility identical to that ofCOS cell-synthesized amino terminally processed form is detected at asubstantial level in the stomach and lung tissue and at a markedly lowerlevel in the forebrain, midbrain, and hindbrain tissues of15.5-day-postcoitum mouse embryos. These tissues have all been shown toexpress Shh RNA. The 19 kDa peptide is not detected in liver or latelimb tissues, which do not express Shh RNA. Thus, the proteolyticprocessing of Shh observed in cell culture also occurs in embryonicmouse tissue.

[0494] The cross-reactivity of the amino terminus-specific mouse Shhantiserum with chicken Shh protein allowed for examination of expressionof Shh in chicken embryonic tissue. The antiserum detects the 19-kDaamino terminally processed form of chicken Shh in transfected COS cells,as well as in two tissues which have been shown by whole-mount in situhybridization and antiserum staining to express high levels of Shh RNAand protein, i.e., the posterior region of the limb bud and the ventralregion of the anterior CNA (Riddle et al. (1993) Cell 75:1401-1416).Therefore, the expected proteolytic processing of Shh occurs in chickenembryonic tissues, and diffusion of the 19-kDa protein does not extendinto the anterior limb buds and dorsal CNS.

[0495] (v) Hedgehog Processing

[0496] In summary, the results discussed above demonstrate that themouse and chick Shh genes encode secreted glycoproteins which undergoadditional proteolytic processing. Data indicate that this processingoccurs in an apparently similar fashion in a variety of cell typessuggesting that it is a general feature of the Shh protein, and notunique to any particular expression system. For mouse Shh, data indicatethat both products of this proteolytic processing are secreted. Theseobservations are summarized in FIG. 13.

[0497] It was observed that the 19 kDa amino peptide accumulates to alower level in the medium than the 27 kDa carboxyl peptide. This mayreflect inefficient secretion or rapid turnover of this species oncesecreted. Alternatively, the smaller form may associate with the cellsurface or extracellular matrix components making it difficult to detectin the medium. The insensitivity of the secreted, larger form to Endo His a common feature of secreted glycoproteins. During transit throughthe Golgi apparatus, the Asn-linked carbohydrate moiety is modified by aseries of specific glycosidases (reviewed in Kornfeld, R. and Kornfeld,S., (1985) Annu. Rev. Biochem 54:631-664; Tarentino, A. L. et al.,(1989) Methods Cell Biol. 32:111-139). These modifications convert thestructure from the immature “high mannose” to the mature “complex” type.At one step in this process, a Golgi enzyme, α-mannosidase II, removestwo mannose residues from the complex rendering it insensitive to Endo H(Kornfeld, R. and Kornfeld, S., (1985) Annu. Rev. Biochem 54:631-664).

[0498] The biochemical behavior of mouse Shh appears to be quite similarto that described for the Drosophila Hedgehog (Dros-HH) protein (Lee, J.L. et al., (1992) Cell 71:33-50; Tabata, T. et al., (1992) Genes & Dev.6:2635-2645). In vitro translation of Drosophila hh mRNA, in thepresence of rhicrosomes, revealed products with molecular massescorresponding to fill length protein, as well as to the product expectedafter cleavage of the predicted internal (Type II) signal peptide (Lee,J. L. et al., (1992) Cell 71:33-50). Interestingly, no additional,processed forms were observed. However, such forms could have beenobscured by breakdown products migrating between 20 and 30 kDa. When anRNA encoding a form of the protein lacking the carboxy-terminal 61 aminoacids was translated, no breakdown products were seen, but there isstill no evidence of the proteolytic processing observed with mouse Shh.A similar phenomenon has been observed in these experiments. A reductionin the extent of proteolytic processing is seen when a mouse Shh proteinlacking 10 carboxy-terminal amino acids is translated in vitro orexpressed in cos cells (data not shown). This suggests that sequences atthe carboxy termini of Hh proteins act at a distance to influence theefficiency of processing.

[0499] Recently, Lee et al. (Science 266:1528-1537, 1994) described thebiochemical behavior of the Drosophila HH protein. Using region-specificantisera, they detected similar processed forms of HH in embryonictissues, thus confirming studies in which processing of HH was observedin embryos forced to express high levels of HH from a heat shockpromoter (Tabata and Kornberg (1994) Cell 76:89-102). Thus, DrosophilaHH is processed to yield a 19 kDa amino-terminal peptide and a 25 kDacarboxy-terminal peptide. Furthermore, Lee et al. concluded that theproduction of the processed forms occurs via an autocatalytic mechanismand identified a conserved histidine residue (at position 329, accordingto Lee et al. (Science 266:1528-1537, 1994)) which is required forself-cleavage of HH protein in vitro and in vivo. The significance ofthe proteolytic processing is demonstrated by the inability ofself-processing-either because of mutation of this histidine residue orbecause of truncation of sequences at the extreme carboxy terminus-tocarry out HH functions in Drosophila embryos.

[0500] Their studies of the biochemical behavior of mouse and chickenShh and mouse Ihh proteins correlate well with the Drosophila studies ofLee et al. (Science 266:1528-1537, 1994) in that the similar proteolyticprocessing of endogenous vertebrate proteins in embryonic tissues wasdemonstrated. Furthermore, it was demonstrated that the efficiency ofprocessing depends on sequences located at the extreme carboxy terminusof mouse Shh. Interestingly, it has also been shown that he specificityof mouse Shh cleavage may depend on targeting of the protein to thesecretory pathway, since a form lacing a signal peptide is processedinto an approximately 28-kDa amino-terminal form. A similar protein isobserved as the predominant species when it was attempted to expressfull-length mouse Shh in bacteria (data no shown). Lee et al. (Science266:1528-1537, 1994) have demonstrated that two zebra fish hedgehogproteins undergo proteolytic processing when translated in vitro, evenin the absence of microsomal membranes. The electrophoretic mobilitiesof the processed peptides are consistent with cleavage occurring at aposition similar to that of the Drosophila HH cleavage site.Furthermore, they showed that the cleavage fails to occur if theconserved histidine residue is mutated, arguing for an autoproteolyticmechanism similar to that of the Drosophila protein. However, theprocessing of mouse or chicken Shh protein translated in vitro was notdetected unless microsomal membrane are included. Therefore, it ispossible that correct proteolytic processing of vertebrate hedgehogproteins is dependent on specific incubation conditions or may requirecellular factors in addition to Shh itself.

[0501] An additional correlation between the work presented here andthat of Lee et al. (Science 266:1528-1537, 1994) concerns the differentbehaviors of the amino (smaller) and carboxy (larger) terminallyprocessed forms of the hedgehog proteins. The evidence is presented thatthe 27 kDa carboxy-terminal form diffuses more readily from expressingcells than the 19 kDa amino-terminal form, which seems to be retainednear the cell surface. The polyanions heparin and suramin appear capableof releasing the amino peptide into the medium. Similarly, theamino-terminal form of Drosophila HH is more closely associated with theRNA expression domain in embryonic segments than is the carboxy-terminalform, and the amino-terminal form binds to heparin agarose beads.Therefore, the distinct behaviors of the different hedgehog peptideshave been conserved across phyla.

[0502] The observed molecular masses of the amino terminally processedforms of mouse and chicken Shh, mouse Ihh proteins, and Drosophila HHare between 19 and 20 kDa. Therefore, the predicted secondaryproteolytic cleavage site would be located near the border of thesequences encoded by the second and third exons. Interestingly, theregion marks the end of the most highly related part of the hedgehogproteins. The amino terminal (smaller) form would contain the mosthighly conserved portion of the protein. In fact, the amino acidsencoded by exons one and two (exclusive of sequences upstream of theputative signal peptide cleavage sites) share 69% identity betweenDrosophila Hh and mouse Shh, and 99% identity between chick and mouseShh. Amino acid identity in the region encoded by the as third exon ismuch lower 30% mouse to Drosophila and 71% mouse to chick (Echelard, Y.et al., (1993) Cell 75:1417-1430).

[0503] However, the boundary between sequences encoded by exons 2 and 3is unlikely to be the actual proteolytic processing site, because aDrosophila HH protein containing a large deletion which extends threeamino acids beyond this boundary is still cleaved at the expectedposition in vitro (Lee et al. (1994) Science 266:1528-1537). Moreover,the analysis of an amino-terminal mouse Shh peptide truncated at aminoacid 193 (the fourth amino acid encoded by exon 3, described below)suggests that normal cleavage must occur downstream of this position.Close examination of hedgehog protein sequences reveals that strongsequence conservation between the Drosophila and vertebrate proteinscontinues for only a short distance into the third exon. If it isassumed that cleavage will generate an amino terminal product of nogreater than 20 kDa, given the resolution of analysis, all of the datawould indicate that cleavage occurs at 1 of the 10 amino acids withinthe mouse Shh positions 194-203, according to Echelard et al. (Cell75:1417-1430, 1993).

[0504] (vi) Hedgehog Signalling

[0505] In order to satisfy the criteria for intercellular signaling,hedgehog proteins must be detected outside of their domains ofexpression. This has been clearly demonstrated for Drosophila HH. Usingan antiserum raised against nearly full length Dros-HH protein, Tabataand Kornberg (Tabata, T. and Kornberg, T. B., (1992) Cell 76:89-102)detect the protein in stripes that are slightly wider than the RNAexpression domains in embryonic segments, and just anterior to theborder of the RNA expression domain in wing imaginal discs. Similarly,Taylor, et. al., (1993) Mech. Dev. 42:89-96, detected HH protein indiscrete patches within cells adjacent to those expressing hh RNA inembryonic segments using an antiserum directed against an amino-terminalportion of Hh which, based on the proteolytic processing data (Tabata,T. et al., (1992) Genes & Dev. 6:2635-2645), is not likely to recognizethe carboxyl cleavage product.

[0506] The detection of Hh beyond cells expressing the hh gene isconsistent with the phenotype of hh mutants. In these animals, cellularpatterning in each embryonic parasegment in disrupted resulting in anabnormal cuticular pattern reminiscent of that seen in wg mutants.Further analysis has revealed that the loss of hh gene function leads toloss of wg expression in a thin stripe of cells just anterior to the hhexpression domain (Ingham, P. W. and Hidalgo, A., (1993) Development117:283-291). This suggests that Hh acts to maintain wg expression inneighboring cells. The observation that ubiquitously expressed h leadsto ectopic activation of wg supports this model (Tabata, T. andKornberg, T. B., (1992) Cell 76:89-102). In addition to these geneticstudies, there is also indirect evidence that Hh acts at a distance fromits site of expression to influence patterning of the epidermis(Heemskerk, J. and DiNardo, S., (1994) Cell 76:449-460).

[0507] The apparent effect of Drosophila Rh on neighboring cells, aswell as on those located at a distance from the site of hh expression isreminiscent of the influence of the notochord and floor plate on thedeveloping vertebrate CNS, and of the ZPA in the limb. The notochord (asite of high level Shh expression) induces the formation of the floorplate in a contact dependent manner, while the notochord and floor plate(another area of strong Shh expression) are both capable of inducingmotorneurons at a distance (Placzek, M. et al., (1993) Development117:205-218; Yamada, T. et al., (1993) Cell 73:673-686).

[0508] Moreover ZPA activity is required not only for patterning cellsin the extreme posterior of the limb bud where Shh is transcribed, butalso a few hundred microns anterior of this zone. Several lines ofevidence indicate that Shh is able to induce floor plate (Echelard, Y.et al., (1993) Cell 75:1417-1430; Roelink, H. et al., (1994) Cell76:761-775) and mediate the signaling activity of the ZPA (Riddle, R. D.et al., (1993) Cell 75:1401-1416). Since it has been shown that Shh iscleaved, it can be speculated that the processed peptides may havedistinct activities. The smaller amino terminal form, which appears tobe more poorly secreted, less stable or retained at the cell surface orin the extracellular matrix, may act locally. In contrast, the largercarboxy terminal peptide could possibly function at a distance. In thisway, Shh peptides may mediate distinct signaling functions in thevertebrate embryo Alternatively, the carboxy-terminal peptide may benecessary only for proteolytic processing, with all signaling activityresiding in the amino-terminal peptide.

EXAMPLE 7 Sonic Hedgehog and Fgf-4 Act Through a Signaling Cascade andFeedback Loop to Integrate Growth and Patterning of the Developing LimbBud

[0509] (i) Experimental Procedures

[0510] Cloning of Chicken Fgf-4 and Bmp-2

[0511] A 246 bp fragment of the chicken Fgf-4 gene was cloned by PCRfrom a stage 22 chicken limb bud library. Degenerate primers weredesigned against previously cloned Fgf-4 and Fgf-6 genes: fgf5′ (sense)AAA AGC TTT AYT GYT AYG TIG GIA THG G (SEQ ID No:38) and fgf3′(antisense) AAG AAT TCT AIG CRT TRT ART TRT TIG G (SEQ ID No:39).Denaturation was at 94° C. for 2 min, followed by 30 cycles of 94° C.for 30 sec, 50° C. for 60 sec, and 72° C. for 30 sec, with a finalextension at 72° C. for 5 min. The PCR product was subdcloned into theBluescript SK+ vector. A clone was sequenced and confirmed as Fgf-4 bycomparison with previously published Fgf-4 genes and a chicken Fgf-4gene sequence kindly provided by Lee Niswander.

[0512] BMP-related sequences were amplified from a stage 22 posteriorlimb bud cDNA library prepared in Bluescript using primers andconditions as described by Basler, et al. (1993). Amplified DNAs werecloned and used to screen a stage 22 limb bud library prepared in λ-Zap(Stratagene). Among the cDNAs isolated was chicken Bmp-2. Its identitywas confirmed by sequence comparison to the published clones (Francis,et al., (1994) Development 120:209-218) and by its expression patternsin chick embryos.

[0513] Chick Surgeries and Recombinant Retroviruses

[0514] All experimental manipulations were performed on White Leghornchick embryos (S-SPF) provided by SPAFAS (Norwich, Conn). Eggs werestaged according to Hamburger and Hamilton (1951) J. Exp. Morph.88:49-92.

[0515] Viral supernatants of Sonic/RCAS-A2 or a variant containing aninfluenza hemaglutinin epitope tag at the carboxyl terminus of thehedgehog protein (Sonic7. 1/RCAS-A2, functionally indistinguishable fromSonic/RCAS-A2), were prepared as described (Hughes, et al., (1987) J.Virol. 61:3004-13; Fekete and Cepko, (1993) Mol. & Cell. Biol.13:2604-13; Riddle, et al., (1993) Cell 75:1401-16). For focalinjections the right wings of stage 18-21 embryos were transientlystained with nile blue sulfate (0.01 mg/ml in Ringer's solution) toreveal the AER. A trace amount of concentrated viral supernatant wasinjected beneath the AER.

[0516] The AER was removed using electrolytically sharpened tungstenwire needles. Some embryos had a heparin-acrylic bead soaked in FGF-4solution (0.8 mg/ml; a gift from Genetics Institute) or PBS stapled tothe limb bud with a piece of 0.025 mm platinum wire (Goodfellow,Cambridge UK) essentially as described by Niswander et al, (1993) Cell75:579-87.

[0517] Limbs which were infected with SonicIRCAS virus after AER removalwere infected over a large portion of the denuded mesoderm to ensuresubstantial infection. Those embryos which received both an Fgf-4 soakedbead and virus were infected only underneath the bead.

[0518] In Situ Hybridizations and Photography

[0519] Single color whole mount in situ hybridizations were performed asdescribed (Riddle, et al., (1993) Cell 75:1401-16). Two color wholemount in situ hybridizations were performed essentially as described byJowett and Lettice (1994) Trends Genet. 10:73-74. The second colordetection was developed using 0.125 mg/ml magenta-phos (Biosynth) as thesubstrate. Radioactive in situ hybridizations on 5 μm sections wasperformed essentially as described by Tessarollo, et al. (1992)Development 115:11-20.

[0520] The following probes were used for whole mount and section insitu hybridizations: Sonic: 1.7 kb fragment of pHH2 (Riddle, et al.,(1993) Cell 75:1401-16). Bmp-2: 1.5 kb fragment encoding the entire openreading frame. Fgf-4: 250 bp fragment described above. Hox d-11: a 600bp fragment, Hoxd-13: 400 bp fragment both including 5′ untranslatedsequences and coding sequences upstream of the homeobox. RCAS: 900 bpSalI-ClaI fragment of RCAS (Hughes et al., (1987) J Virol. 61:3004-12).

[0521] (ii) Relationship of Sonic to Endogenous Bmp-2 and Hoxd GeneExpression

[0522] The best candidates for genes regulated by Sonic in vivo are thedistal members of the Hoxd gene cluster, Hoxd-9 through -13, and Bmp-2.Therefore, the relationships of the expression domains of these genes ina staged series of normal chick embryos were analyzed. Hoxd-9 andHoxd-10 are expressed throughout the presumptive wing field at stage 16(Hamburger and Hamilton, (1951) J. Exp. Morph. 88:49-92), prior to thefirst detectable expression of Sonic at early stage 18. Hoxd-11expression is first detectable at early stage 18, the same time asSonic, in a domain coextensive with Sonic. Expression of Hoxd-12 andHoxd-13 commence shortly thereafter. These results suggest that Sonicmight normally induce, directly or indirectly, the expression of onlythe latter three members of the cluster, even though all five are nestedwithin the early limb bud.

[0523] As limb outgrowth proceeds Sonic expression remains at theposterior margin of the bud. In contrast the Hoxd gene expressiondomains, which are initially nested posteriorly around the Sonic domain,are very dynamic and lose their concentric character. By stage 23 theHoxd-11 domain extends anteriorly and distally far beyond that of Sonic,while Hoxd-13 expression becomes biased distally and displaced fromSonic.

[0524] While it is not clear whether Bmp-2 is expressed before Sonic(see Francis et. al., (1994) Development 120:209-218) Bmp-2 is expressedin a mesodermal domain which apparently overlaps and surrounds that ofSonic at the earliest stages of Sonic expression As the limb buddevelops, the mesodermal expression of Bmp-2 remains near the posteriorlimb margin, centered around that of Sonic, but in a larger domain thanSonic. This correspondence between Sonic and Bmp-2 expression lastsuntil around stage 25, much longer than the correspondence between Sonicand Hoxd gene expression. After stage 25 Bmp-2 expression shiftsdistally and is no longer centered on Sonic.

[0525] (iii) Relationship of Sonic to Induced Bmp-2 and Hoxd GeneExpression

[0526] The fact that the expression domains of the Hoxd genes divergeover time from that of Sonic hedgehog implies that Sonic does notdirectly regulate their later patterns of expression. This does notpreclude the possibility that the later expression domains aregenetically downstream of Sonic. If this were the case, exogenouslyexpressed Sonic would be expected to initiate a program of Hoxd geneexpression which recapitulates that seen endogenously. Therefore, thespatial distribution of Hoxd gene expression at various times followingSonic misexpression was compared. The anterior marginal mesoderm ofearly bud (Stage 18-20) wings was injected at a single point under theAER with a replication competent virus that expresses a chicken SoniccDNA. Ectopic Sonic expressed by this protocol leads to both anteriormesodermal outgrowth and anterior extension of the AFR.

[0527] The Sonic and Hoxd gene expression domains in the infected limbswere analyzed in sectioned and intact embryos. Viral Sonic message isfirst detected approximately 18 hours after infection at the anteriormargin, at the same time as, and approximately coextensively with,induced Hoxd-11. This suggests that Sonic can rapidly induce Hoxd-11expression and that the lag after injection represents the time requiredto achieve Sonic expression. By 35 hours post infection distal outgrowthof infected cells combined with lateral viral spread within theproliferating cells leads to viral expression in a wedge which isbroadest at the distal margin and tapers proximally. By this time,Hoxd-11 expression has expanded both antero-proximally and distally withrespect to the wedge of Sonic-expressing cells, into a domain whichappears to mirror the more distal aspects of the endogenous Hoxd-11domain. Weak Hoxd-13 expression is also detected at 35 hours in a subsetof the Sonic expressing domain at its distal margin. 51 hours afterinfection the relationship of Sonic and Hoxd-11 expression is similar tothat seen at 35 hours, while the induced Hoxd-13 expression has reachedwild type levels restricted to the distal portions of the ectopicgrowth. Thus the ectopic Hoxd expression domains better reflect theendogenous patterns of expression than they do the region expressingSonic. This suggests that there are multiple factors regulating Hoxdexpression but their actions lie downstream of Sonic.

[0528] Since the endogenous Bmp-2 expression domain correlates well withthat of Sonic, and Bmp-2 is induced by ZPA grafts, it was looked to seeif Bmp-2 is also induced by Sonic. Bmp-2 is normally expressed in twoplaces in the early limb bud, in the posterior mesoderm and throughoutthe AER (Francis, et al., (1994) Development 120:209-218). In injectedlimb buds additional Bmp-2 expression is seen in both the anteriormesoderm and in the anteriorly extended AER. The domain of Bmp-2expression is slightly more restricted than that of viral expression,suggesting a delay in Bmp-2 induction. Bmp-2 expression in both themesoderm and ectoderm is thus a downstream target of Sonic activity inthe mesoderm. In contrast to the expression domains of the Hoxd genes,the endogenous and ectopic Bmp-2 expression domains correlate well withthat of Sonic. This suggests that Bmp-2 expression is regulated moredirectly by Sonic than is expression of the Hoxd genes.

[0529] (iv) The AER and Competence to Respond to Sonic

[0530] Ectopic activation of Hoxd gene expression is biased distally invirally infected regions, suggesting that ectodermal factors, possiblyfrom the AER, are required for Hoxd gene induction by Sonic. To testthis, Sonic virus was injected into the proximal, medial mesoderm ofstage 21 limb buds, presumably beyond the influence of the AER. Althoughthe level of Sonic expression was comparable to that observed in distalinjections, proximal misexpression of Sonic did not result in ectopicinduction of the Hoxd genes or Bmp-2, nor did it result in any obviousmorphological effect (data not shown). The lack of gene inductionfollowing proximal misexpression of Sonic suggests that exposure toSonic alone is insufficient to induce expression of these genes.

[0531] This was tested more rigorously by injection of Sonic virus intothe anterior marginal mesoderm of stage 20/21 limb buds after theanterior half of the AER had been surgically removed. Embryos wereallowed to develop for a further 36 to 48 hours before harvesting.During this time the AER remaining on the posterior half of the limb budpromotes almost wild type outgrowth and patterning of the bud. Geneexpression was monitored both in sectioned and intact embryos. In thepresence of the AFR, Sonic induces both anterior mesodermalproliferation and expression of Hoxd-11, Hoxd-13 and Bmp-2. In theabsence of the overlying AER, Sonic does not induce either mesodermalproliferation or expression of these genes above background. Signalsfrom the AER are thus required to allow both the proliferative andpatterning effects of Sonic on the mesoderm.

[0532] Since application of FGF protein can rescue other functions ofthe AER such as promoting PD outgrowth and patterning, it was sought todetermine whether FGFs might also promote mesodermal competence torespond to Sonic. FGF-4-soaked beads were stapled to AER-denudedanterior mesoderm which was infected with Sonic virus. Gene expressionand mesodermal outgrowth were monitored as described previously. In thepresence of both Sonic virus and FGF-4 protein, Hoxd-11, Hoxd-13 andBmp-2 expression are all induced. The expression levels of the inducedgenes are similar to or greater than the endogenous expression levels,and are equivalent in magnitude to their induction in the presence ofthe AER. Thus Fgf-4 can induce the competence of the mesoderm to respondto Sonic.

[0533] Sonic alone is insufficient to induce either gene expression ormesodermal proliferation in the absence of the AER, while thecombination of Sonic and FGF-4 induces both proliferation and geneexpression. It was than asked whether FGF-4 alone has any effect on geneinduction or mesodermal proliferation. Application of FGF-4 in theabsence of Sonic virus does not induce Hoxd or Bmp-2 gene expressionabove control levels, however FGF-4 alone induces mesodermal outgrowth.These results suggest that mesodermal gene activation requires directaction of Sonic on the mesoderm and that proliferative response to Sonicis indirect, due to the induction of FGFs.

[0534] (v) Sonic Induces Polarized Fgf-4 Expression in the AER

[0535] Fgf-4 is expressed in a graded fashion in the AER of the mouselimb bud, with maximal expression at the posterior region of the AERtapering to undetectable levels in the anterior ridge (Niswander andMartin, (1992) Development 114:755-68). Therefore, it was appropriate toinvestigate whether Fgf-4 is asymmetrically expressed in the chick AER,and whether its expression is induced by Sonic. A fragment of thechicken Fgf-4 gene was cloned from a stage 22 chicken limb library byPCR using degenerate primers designed from mouse Fgf-4 and Xenopus e-Fgfsequence; based on information provided by L. Niswander and G. Martin.Assignment of gene identity was based on primary sequence as well ascomparison of expression patterns with that of murine Fgf-4 (Niswanderand Martin, (1992) Development 114:755-68). Whole mount in situhybridization analysis showed strong limb expression of chick Fgf-4 inthe AER. Fgf-4, like Bmp-2, is expressed all the way to the posteriorborder of the AER, but its anterior domain ends before the morphologicalend of the AER creating a posterior bias that has also been observed byNiswander et al., (1994) Nature (in press). Expression is first detectedin the distal AER at about stage 18. As outgrowth proceeds the posteriorbias develops. Expression peaks around stage 24/25 and then fades bystage 28/29.

[0536] The expression domain of Fgf-4 becomes posteriorly biased asSonic is expressed in the posterior mesoderm. This observation isconsistent with Sonic influencing the expression of Fgf-4 in theposterior AER. To test the effect of Sonic on Fgf-4 expression in theAER, stage 18-20 embryos were infected with Sonic virus in a singlepoint at their anterior margin beyond the anterior limit of the AER. Theembryos were harvested one to two days later, when an extension of theanterior AER became apparent. The expression of Fgf-4 was analyzed by insitu hybridization. Fgf-4 expression is induced in the anteriormostsegment of the AER, in a region which is discontinuous with theendogenous expression domain, and overlies the domain of viral Sonicinfection. This result contrasts with the Bmp-2 expression induced inthe extended AER, which is always continuous with the endogenousexpression domain. The asymmetry of the induced Fgf-4 expressionindicates that Sonic polarizes the extended AER, much as a ZPA graftdoes (Maccabe and Parker, (1979) J. Embryol. Exp. Morph. 53:67-73).Since FGFs by themselves are mitogenic for limb mesoderm, these resultsare most consistent with Sonic inducing distal proliferation indirectly,through the induction of mitogens in the overlying AER.

[0537] (vi) Reciprocal Regulation of Sonic by Fgf-4

[0538] Sonic thus appears to be upstream of Fgf-4 expression in the AER.However, since the AER is required to maintain polarizing activity inthe posterior mesoderm (Vogel and Tickle, (1993) Development 19:199-206;Niswander et al., (1993) Cell 75:579-87), Sonic may also be downstreamof the AER. If Sonic is regulated by the AER and the AER by Sonic, thiswould imply that they are reinforcing one another through a positivefeedback loop.

[0539] To test whether the AER dependence of ZPA activity is controlledat the level of transcription of the Sonic gene, Sonic expressionfollowing removal of the AER from the posterior half of the limb bud wasassayed. Sonic expression is reduced in an operated limb compared to thecontralateral control limb within ten hours of AER removal, indicatingthat Sonic expression is indeed AER dependent. The dependence of Sonicexpression on signals from the AER suggests that one of the functions ofthe AER is to constrain Sonic expression to the more distal regions ofthe posterior mesoderm.

[0540] In addition to their mitogenic and competence-inducingproperties, FGFs can also substitute for the AER to maintain the ZPA. Inorder to test whether FGFs can support the expression of Sonic, beadssoaked in FGF-4 protein were stapled to the posterior-distal tips oflimb buds after posterior AER removal. Embryos were assayed for Sonicexpression approximately 24 hours later, when Sonic expression isgreatly reduced in operated limb buds which had not received an FGF-4bead. Strong Sonic expression is detectable in the posterior mesoderm,slightly proximal to the bead implant, and reflecting the normal domainof Sonic expression seen in the contralateral limb. With the findingthat FGF-4 can maintain Sonic expression, the elements required for apositive feedback loop between Sonic expression in the posteriormesoderm and Fgf-4 expression in the posterior AER are established (seealso Niswander et al. (1994) Nature (in press)).

[0541] The induction of Bmp-2 expression by Sonic requires signals fromthe AER, and its domain correlates over time with that of Sonic.Therefore, it was interesting to learn if the continued expression ofBmp-2 also requires signals from the AER, and if so, whether they couldbe replaced by FGF-4. To test this, Bmp-2 expression following posteriorAER removal, and following its substitution with an FGF-4 bead wasassayed. Bmp-2 expression fades within hours of AER removal, and can berescued by FGF-4. These data indicate that the maintenance of Bmp-2expression in the posterior mesoderm, like that of Sonic, is dependenton signals from the AER, which are likely to be FGFs.

[0542] (vii) The Mesodermal Response to Sonic

[0543] It has been found that only mesoderm underlying the AER isresponsive to Sonic, apparently because the AER is required to providecompetence signals to the limb mesoderm. Fgf-4, which is expressed inthe AER, can substitute for the AER in this regard, and thus might actin combination with Sonic to promote Hoxd and Bmp-2 gene expression inthe mesoderm. FGFs may be permissive factors in a number of instructivepathways, as they are also required for activins to pattern Xenopusaxial mesoderm (Cornell and Kimelman, (1994) Development 120:2187-2198;LaBonne and Whitman, (1994) Development 120:463-472).

[0544] The induction of Hoxd and Bmp-2 expression in response to Sonicand FGF-4 in the absence of an AER suggests that the mesoderm is adirect target tissue of Sonic protein. Since Sonic can induce Fgf-4expression in the AER, it follows that Sonic also acts indirectly on themesoderm through the induction of competence factors in the AER.

[0545] (viii) Downstream Targets and a Cascade of Signals Induced bySonic

[0546] The five AbdB-like Hoxd genes, Hoxd-9 through -13, are initiallyexpressed in a nested pattern centered on the posterior of the limb bud,a pattern which suggests they might be controlled by a common mechanism(Dolle, et al., (1989) Cell 75:431-441; Izpisua-Belmonte, et al., (1991)Nature 350:585-9). The analysis of the endogenous and induced domains ofHoxd gene expression suggests that Sonic normally induces expression ofHoxd-11, -12 and -13. In contrast it was found that Hoxd-9 and -10expression initiate before Sonic mRNA is detectable. This implies thatat least two distinct mechanisms control the initiation of Hoxd geneexpression in the wing bud, only one of which is dependent on Sonic.

[0547] Several observations suggest that the elaboration of the Hoxdexpression domains is not controlled directly by Sonic, but rather bysignals which are downstream of Sonic. The Hoxd expression domainsrapidly diverge from Sonic, and evolve into several distinct subdomains.Moreover these subdomains appear to be separately regulated, as analysisof the murine Hoxd-11 gene promoter suggests that it containsindependent posterior and distal elements (Gerard, et al., (1993) Embo.J. 12:3539-50). In addition, although initiation of Hoxd-11 through -13gene expression is dependent on the AER, their expression is maintainedfollowing AER removal (Izpisua-Belmonte, et al., (1992) Embo. J.11:1451-7). As Sonic expression fades rapidly under similar conditions,this implies that maintenance of Hoxd gene expression is independent ofSonic. Since ectopic Sonic can induce a recapitulation of the Hoxdexpression domains in the limb, it can be concluded that althoughindirect effectors appear to regulate the proper patterning of the Hoxdexpression domains, they are downstream of Sonic. Potential mediators ofthese indirect effects include Bmp-2 in the mesoderm and Fgf-4 from theAER.

[0548] In contrast to the Hoxd genes, Bmp-2 gene expression in theposterior limb mesoderm appears to be continually regulated by Sonic. Itwas found that both endogenous and ectopic Bmp-2 expression correspondto that of Sonic. Furthermore, continued Bmp-2 expression is dependenton the AER and can be rescued by FGF-4. It is likely that this is anindirect consequence of the fact that Sonic expression is alsomaintained by the AER and can be rescued by FGF-4. In fact, Bmp-2expression might be a direct response of cells to secreted Sonicprotein. The differences between Bmp-2 and Hoxd gene expression suggestthat multiple pathways downstream of Sonic regulate gene expression inthe mesoderm.

[0549] Bmp-2 itself is a candidate for a secondary signaling molecule inthe cascade of patterning events induced by Sonic. Bmp-2 is a secretedmolecule of the TGF-β family and its expression can be induced by Sonic.This appears to be an evolutionarily conserved pathway, as HH, theDrosophila homolog of Sonic, activates the expression of dpp, thehomolog of Bmp-2, in the eye and wing imaginal discs (Heberlein, et al.,(1993) Cell 75:913-26; Ma, et al., (1993) Cell 75:927-38; Tabata andKornberg, (1994) Cell 76:89-102). Expression of HH is normally confinedto the posterior of the wing disc. Ectopic expression of HH in theanterior of the disc results in ectopic expression of dpp and ultimatelyin the duplication of wing structure with mirror image symmetry (Basslerand Struhl, (1994) Nature 368:208-214). This effect is strikinglyparallel to the phenotypic results of ectopic expression of Sonic in thechick limb.

[0550] (ix) Regulation of Sonic Expression

[0551] Sonic expression is activated in the posterior of the limb budvery early during mesodermal outgrowth (Riddle et al., (1993) Cell75:1401-16). The factors which initiate this localized expression arenot yet identified but ectopic expression of Hoxb-8 at the anteriormargin of the mouse limb bud results in the activation of a seconddomain of Sonic expression under the anterior AER (Charité el al.,(1994) Cell 78:589-601). Since retinoic acid is known to be able toinduce the expression of Hoxb-8 and other Hox genes in vitro (Mavilio etal., (1988) Differentiation 37:73-79) it is possible that endogenousretinoic acid acts to make cells competent to express Sonic by inducingexpression of upstream Hox genes, either in the very early limb bud orin the flank prior to the limb bud formation.

[0552] Several lines of evidence suggest that once induced Sonicexpression is dependent on signals from the posterior AER. Following itsinitiation in the posterior limb mesoderm, the Sonic expression domainmoves distally as the limb bud grows out, always remaining subjacent tothe AER. Similarly, Sonic expression can also be induced on the anteriormargin of the limb bud by implantation of a retinoic acid bead, but theinduced ectopic expression is limited to the mesoderm directlyunderlying the AER (Riddle, et al., (1993) Cell 75:1401-16). Inaddition, ZPA activity fades rapidly following removal of the AER(Niswander, et al., (1993) Cell 75:579-87; Vogel and Tickle, (1993)Development 119:199-206), and ZPA grafts only function when placed inclose proximity to the AER (Tabin, (1991) Cell 66:199-217; Tickle,(1991) Development Supp. 1:113-21). The observation that continued Sonicexpression depends on signals from the posterior AER reveals themechanism underlying these observations.

[0553] The reliance of Sonic expression on AER-derived signals suggestsan explanation for the distal shift in Sonic expression during limbdevelopment (Riddle et al., (1993) Cell 75:1401-16). Signals from theAER also promote distal outgrowth of the mesodermal cells of theprogress zone, which in turn results in the distal displacement of theAER. Hence, as maintenance of Sonic expression requires signals from theAER, its expression domain will be similarly displaced.

[0554] It was found that replacement of the AER with FGF-4 soaked beadsresults in the maintenance of Sonic expression. This result isconsistent with the previous findings that ZPA activity can bemaintained in vivo and in vitro by members of the FGF family (Anderson,et al., (1993) Development 117:1421-33; Niswander et al., (1993) Cell75:1401-16; Vogel and Tickle, (1993) Development 119:199-206). SinceFgf-4 is normally expressed in the posterior AER, these results suggestthat Fgf-4 is the signal from the ectoderm involved in maintaining Sonicexpression.

[0555] (x) Sonic and Regulation and Maintenance of the AER

[0556] Sonic can induce anterior extensions of the AER which have aninverted polarity relative to the endogenous AER. This polarity isdemonstrated by examining the expression of two markers in the AER. Innormal limbs Bmp-2 is expressed throughout the AER, while Fgf-4 isexpressed in the posterior two thirds of the AER. In the extended AERresulting from ectopic Sonic expression, Bmp-2 is again found throughoutthe AER, while Fgf-4 expression is biphasic, found at either end of theAER, overlying the anterior and posterior mesodermal domains expressingSonic. These results are consistent with previous observations thatantero-posterior polarity of the AER appears to be regulated by theunderlying mesoderm, and that ZPA grafts lead to the induction ofectopic, polarized AER tissue (Maccabe and Parker, (1979) J. Embryol.Exp. Morph. 53:67-73). Our results also suggest that the normal APpolarity of the AER is a reflection of endogenous Sonic expression. Theinduced AER is sufficient to promote complete PD outgrowth of theinduced structures (Riddle et al., (1993) Cell 75:1401-16). Hencewhatever factors are necessary to maintain the AER are also downstreamof Sonic.

[0557] (xi) A Positive Feedback Loop Between Sonic and Fgf-4

[0558] The induction of Fgf-4 expression by Sonic in the ectopic AER,and the maintenance of Sonic expression by FGF-4 suggest that Sonic andFgf-4 expression are normally sustained by a positive feedback loop.Such a feedback loop would allow the coordination of mesodermaloutgrowth and patterning. This coordination is possible because Sonicpatterns mesodermal tissue and regulates Fgf-4 expression, while FGF-4protein induces mesodermal proliferation and maintains Sonic expression.Moreover mesodermal tissue can only be patterned by Sonic in the contextof a competence activity provided by F8f-4. Thus patterning is alwayscoincident with proliferation.

[0559] It remains possible that exogenously applied Fgf-4 might bemimicking the activity of a different member of the FGF family. Forexample, Fgf-2 is expressed in the limb mesoderm and the AER (Savage etal., (1993) Development Dynamics 198:159-70) and has similar effects onlimb tissue as Fgf-4 (Niswander and Martin, (1993) Nature 361:68-71;Niswander, et al., (1993) Cell 75:579-87; Riley, et al., (1993)Development 118:95-104; Fallon, et al., (1994) Science 264:104-7).

[0560] (xii) Coordinated Regulation of Limb Outgrowth and Patterning

[0561] Patterning and outgrowth of the developing limb are known to beregulated by two major signaling centers, the ZPA and AER. Theidentification of Sonic and FGFs as molecular mediators of theactivities of the ZPA and AER has allowed for dissociation of theactivities of these signaling centers from their regulation, andinvestigation of the signaling pathways through which they function.

[0562] The results presented above suggest that the ability of cells torespond to Sonic protein is dependent on FGFs produced by the AER. Itwas also found that Sonic induces a cascade of secondary signalsinvolved in regulating mesodermal gene expression patterns. In additionevidence was found for a positive feedback loop initiated by Sonic,which maintains expression of Sonic in the posterior mesoderm and Fgf-4in the AER. The feedback loop described suggests a mechanism wherebyoutgrowth and patterning along the AP and PD axes of the limb can becoordinately regulated.

[0563] The results described above further suggest that Sonic acts as ashort range signal which triggers a cascade of secondary signals whoseinterplay determines the resultant pattern of structures. The datasuggest a number of inductive pathways that can be combined to generatea model (FIG. 14) which describes how Sonic, in coordination with theAER, acts to pattern mesodermal tissues along the anterior-posteriorlimb axis, while simultaneously regulating proximal-distal outgrowth.

[0564] Following its induction, Sonic signals to both the limb ectodermand mesoderm. Sonic imposes a distinct polarity on the forming AER,including the posteriorly biased expression of Fg-4, and the AER becomesdependent on continued Sonic expression. The mesoderm, as long as it isreceiving permissive signals from the overlying ectoderm, responds tothe Sonic signal by expressing secondary signaling molecules such asBmp-2 and by activating Hoxd genes. Bmp-2 expression is directlydependent on continued Sonic expression, while the continued expressionof the Hoxd genes, rapidly becomes Sonic. independent. In a reciprocalfashion, maintenance of Sonic hedgehog expression in the posteriormesoderm becomes dependent on signals from the AER. Since the factorsexpressed by the AER are not only required for the maintenance of Sonicexpression and activity, but are also mitogenic, growth and patterningbecome inextricably linked. Coordination of limb development throughinterdependent signaling centers forces the AP and PD structures to beinduced and patterned in tandem. The pathways elucidated herein thusprovide a molecular framework for the controls governing limb patterning

EXAMPLE 8 Sonic, BMP-4, and Hox Gene Expression Suggest a ConservedPathway in Patterning the Vertebrate and Drosophila Gut

[0565] (i) Experimental Procedure

[0566] In Situ Hybridization and Photography

[0567] BMP probes were isolated using primers designed to amplifymembers of the TGF- and BMP families (Basler, K. et al., (1993) Cell73:687-702, eight independent 120 bp BMP fragments were amplified from astage 22 chicken posterior limb bud plasmid cDNA library. Thesefragments were pooled and used to screen an unamplified stage 22 limbbud lambda zap cDNA library constructed as in Riddle et al., (1993) Cell75:1401-16 . Among the BMP related clones isolated were an approximately1.9 kb cDNA clone corresponding to chicken BMP-2 and an approximately1.5 kb cDNA clone corresponding to chicken BMP-4. Both clones containthe entire coding regions. The Sonic clone was obtained as described inRiddle et al, (1993) Cell 75:1401-16. Digoxigenin-UTP labeled RNA probeswere transcribed as per Riddle et al., (1993) Cell 75:1401-16. Briefly,harvested chick embryos were fixed overnight in 4% paraformaldehyde,washed in PBS then processed for whole mount in situ hybridizationmethods are per Riddle et al., (1993)Cell 75:1401-16. Embryos werephotographed from either ventral or dorsal surfaces under transmittedlight using a Nikon zoom stereo microscope with Kodak Ektar 100 ASAfilm. Whole mount in situ hybridization embryos and viscera wereprocessed for sectioning as described in Riddle et al., (1993)Cell75:1401-16. 15-25 μm transverse sections were air dried and photographedwith brightfield or numarski optics using a Zeiss Axiophot microscopeand Kodak Ektar 25 ASA film.

[0568] Chick Embryos and Recombinant Retroviruses

[0569] A retroviral vector engineered to express a full length cDNA ofchicken Sonic, as in Riddle et al. (1993) Cell 75:1401-16, was injectedunilaterally into stage 8-13 chicken embryos targeting the definitiveendoderm at the mid-embryo level. At this stage the CIP has not formedand neither Sonic nor BMP-4 are expressed in the region injectedInjections were performed on the ventral surface on embryos culturedwith their ventral surface facing up (New, D.A.T. (1955) Embryol. Exp.Morph. 3:320-31. Embryos were harvested 18-28 hours after injection andprepared for whole mount in situ hybridization (see above description ofin situ experiment), hybridized with Sonic or BMP-4 digoxigenin labeledprobes.

[0570] In Situ Hybridization with Hox Genes

[0571] Cloned cDNA of the chicken homologues of Hoxa-9,-10,-11,-13; b-9,c-9,-10,-11; d-9,-10,-11,-12, and -13 were used to transcribedigoxigenen-UTP labeled riboprobes for whole mount in situhybridization. Domestic chick embryos were harvested into PBS andeviscerated. The visceral organ block was fixed in 4% paraformaldehydeovernight and processed for whole mount in situ hybridization. Methodsand photographic technique as described above.

[0572] (ii) Expression of Sonic and BMP-4 in Stage 13 Chick EmbryosDetermined by Whole Mount In Situ Hybridization

[0573] Chick gut morphogenesis begins at stage 8 (Hamberger andHamilton, (1987) Nutr. 6:14-23 with a ventral in-folding of the anteriordefinitive endoderm to form the anterior intestinal portal (AIP)(Romanoff, A. L., (1960) The Avian Embryo, The Macmillan Co., NY. Thislengthens posteriorly forming the foregut. A second wave of endodermalinvagination is initiated posteriorly at stage 13, creating the caudalintestinal portal (CIP). The CIP extends anteriorly forming the hindgut.Sonic expression, previously noted in the endoderm of the vertebrate gut(Riddle et al., (1993) Cell 75:1401-16; Echelard et al., (1993) Cell75:1417-1430), is expressed early in a restricted pattern in theendodermal lips of the AIP and CIP. Sonic expression is detected in theendoderm of the AIP and CIP in pre gut closure stages. At later stages,stage 28 embryos, Sonic is expressed in the gut in all levels (fore-,mid-, and hind-gut) restricted to the endoderm. Sonic is known to be animportant inductive signal in other regions of the embryo including thelimb bud (Riddle et al., (1993) Cell 75:1401-16) and neural tube(Echelard et al., (1993) Cell 75:1417-1430; Kraus et al., (1994) Cell75:1437-1444; Roelink et al., (1994) Cell 76:761-775). Since primitivegut endoderm is known to cause gut-specific mesodermal differentiationwhen combined with non-gut mesenchyme (Haffen et al., (1987) Nutr.6:14-23), we speculated that Sonic might function as an inductive signalto the visceral mesoderm. A potential target gene for the action ofSonic was suggested by analogy to the Drosophila imaginal discs whereHH, the homologue of vertebrate Sonic, activates the expression of theTGF-β related gene dpp in adjacent cells (Tabata abd Kornberg, (1994)Cell 76:89-102; Heberlein et al., (1993) Cell 75:913-926; Ma et al.,(1993) Cell 75:913-926; Basler et al., (1993) Cell 73:687-702). Thereare two vertebrate homologues of dpp, BMP-2 and BMP-4. The earliestdetectable expression of BMP-4 occurs simultaneously with the firstobservable expression of Sonic in the developing gut. BMP-4 is expressedin a domain abutting Sonic at the AIP and the CIP, but is restricted tothe adjacent ventral mesoderm. BMP-4 gut expression persists into laterstage embryos, stage 33 embryos, in the visceral mesoderm only. Thetissue restricted expression of both genes is maintained in all stagesstudied. BMP-2 is not expressed in the gut at the AIP or CIP, but isexpressed in clusters of cells in the gut mesoderm in later stages, apattern distinct from that of BMP-4.

[0574] (iii) Ectopic Expression of Sonic Induces Ectopic Expression ofBMP-4 in Mesodermal Tissues of the Developing Chick

[0575] To test whether Sonic is capable of inducing BMP-4 in themesoderm we an ectopic expression system previously used to study therole of Sonic in limb development was utilized (Riddle et al., (1993)Cell 75:1401-16). A replication competent retrovirus engineered toexpress Sonic was injected unilaterally into the presumptive endodermand visceral mesoderm at mid-embryo positions in stage 8-13 chickembryos in vitro (New, D.A.T. (1955) Embryol. Exp. Morph. 3:320-321).When embryos were examined by in situ hybridization 18-26 hours later,the normal wild type expression of Sonic is detected at the AIP, CIP,and in the midline (neural tube and notochord). Ectopic Sonic expressionis present unilaterally on the left ventral surface. Also, wild typeSonic expression is seen in the floor plate of the neural tube andnotochord. Ectopic expression is seen unilaterally in the visceralendoderm, its underlying splanchnic mesoderm, and somatic mesoderm.BMP-4 expression can be seen induced in the mesoderm at the site ofinjection, in addition to its normal expression in the mesoderm of theCIP. Wild type BMP-4 expression is seen in the most dorsal aspects ofthe neural tube and symmetrical lateral regions adjacent to the neuraltube. Induced BMP-4 expression is present unilaterally in the splanchnicmesoderm at the site of Sonic viral injection, and not in the visceralendoderm.

[0576] Since BMP-4 is, itself, a secreted protein, it could function asa secondary signal in an inductive cascade, similar to the signalcascades from HH to dpp in Drosophila imaginal discs (Tabata abdKornberg, (1994) Cell 76:89-102; Heberlein et al., (1993) Cell75:913-926; Ma et al., (1993) Cell 75:913-926; Basler et al., (1993)Cell 73:687-702) and from Sonic to BMP-2 in the limb bud. In the gut,BMP-4 could act as a secondary signal either as part of a feedback loopto the endoderm or within the visceral mesoderm. This latter possibilityis consistent with the finding that in mice homozygous for a deletion inthe BMP-4 gene, the ventral mesoderm fails to close.

[0577] (iv) Expression of Hox Genes in the Developing Chick Gut

[0578] There is a striking parallel between the apparent role of Sonicas an endoderm-to-mesoderm signal in early vertebrate gut morphogenesisand that of its Drosophila homologue, HH. HH (like Sonic) is expressedin the Drosophila gut endodern from the earliest stages of morphogenesis(Taylor et al., (1993) Mech. Dev. 42:89-96). Its putative receptor,patched, is found in the visceral mesoderm implicating HH (like Sonic)in endodermal-mesodermal inductive interactions. This led toconsideration whether other genes known to be involved in regulatingDrosophila gut development might also play a role in regulating chickgut morphogenesis. Regionally specific pattern in Drosophila gutendoderm is regulated by a pathway involving restricted expression ofhomeotic genes in the mesoderm (McGinnis and Krumlauf, (1992) Cell68:283-302). Although the basis for patterning the vertebrate gut ispoorly understood, in several other regions of the embryo Hox genes havebeen implicated as key regulators of patterns. Vertebrate Hox genes areexpressed in overlapping anteroposterior domains which correlate withstructural boundaries in the developing hindbrain, vertebrae, and limbs(McGinnis and Krumlauf, (1992) Cell 68:283-302). Whole mount in situhybridization was used to test whether these genes are also expressed inthe developing vertebrate hindgut and whether their domains ofexpression correlate with morphologic borders of the chick gut.

[0579] Lumenal gut differentiation creates three morphologically andphysiologically distinct regions: fore-, mid-, and hind-gut. Thefore-gut and hind-gut are the derivatives of the primitive gut tubesinitiated at the AIP and CIP respectively. Ultimately these tubes meetand fuse at the yolk stalk around stage 24-28. The midgut is formed fromboth foregut and hindgut primordia, just anterior and posterior to theyolk stalk.

[0580] The most posterior derivative of the hindgut is the cloaca, thecommon gut-urogenital opening. The rest of the hindgut develops into thelarge intestine. The midgut/hindgut border is demarcated by a pairedtubal structure, the ceca (analogous to the mammalian appendix), whichforms as budding expansions at the midgut/hindgut border at stage 19-20.Anterior to the ceca, the midgut forms the small intestine.

[0581] The expression pattern of the 5′ members of the Hox gene clustersin the chick hindgut by whole mount in situ hybridization was studied.Hox gene expression patterns in the gut are dynamic. They are initiallyexpressed (by stage 10) in broad mesodermal domains extending anteriorlyand laterally. Later they become restricted. By stage 25, the Abd-B likegenes of the Hoxa and Hoxd cluster are regionally restricted in theirexpression in hindgut mesoderm. The most anteriorly expressed gene,Hoxa-9, has an anterior border of expression within the mesoderm of thedistal midgut (to a point approximating the distal third of the midgutlength). Each successive gene within the A and D Hox clusters has a moreposterior domain of expression. Hoxa-10, Hoxd-9 and Hoxd-10 arerestricted in their expression to the ceca. Hoxa-11 and Hoxd-11 have ananterior limit of expression in the mid-ceca at the approximatemidgut/hindgut boundary (Romanoff, A. L. (1960) The Avian Embryo, TheMacmillan Co. NY). Hoxd-12 has an anterior limit at the posterior borderof the ceca and extends posteriorly throughout the hindgut to thecloaca. Hoxa-13 and Hoxd-13 are expressed in the most posteriorlyrestricted domain, in the ventral mesoderm surrounding the cloaca.Hoxa-13 and Hoxd-13 are the only Abd-B like genes which are alsoexpressed within the gut endoderm, from the ceca to the cloaca.

[0582] The only member of the B or C Hox clusters which we found to beexpressed in the hindgut is Hoxc-9. The expression of Hoxc-9 overlapswith its paralogues Hoxa-9 and Hoxd-9 in the midgut mesoderm, but has asharp posterior boundary, complementary to Hoxa-11 and Hoxd-11 in themid-ceca.

[0583] The restricted expression of the Abd-B like Hox genes appear todemarcate the successive regions of the gut which will form the cloaca,the large intestine, the ceca, the mid-ceca at the midgut/hindgutborder, and the lower portion of the midgut (perhaps identifying thatportion of the midgut derived from the posterior gut tube3). Moreover,these molecular events presage regional distinctions. Expression of allHox genes could be detected by stage 14, well before the hindgut lumenis closed (by stage 28) and is maintained in subsequent stages studied.Cytodifferentiation of the hindgut mesoderm and epithelium begins later,at stages 29-31 (Romanoff, A. L. (1960) The Avian Embryo, The MacmillanCo. NY).

[0584] These results suggest that specific Hox genes might beresponsible for regulating morphogenesis of the gut. Consistent withthis, there is an apparent homeotic alteration in the gut of atransgenic mouse in which the anterior limit of expression of Hoxc-8 isshifted rostrally: a portion of foregut epithelium mis-differentiates asmidgut (Pollock and Bieberich, (1992) Cell 71:911-923).

[0585] (v) Conservation in the Expression of Regulatory Genes Involvedin the Formation of Vertebrate and Drosophila Gut

[0586] There is an intriguing parallel between the expression patternsof Sonic, BMP-4, and the Hox genes in the vertebrate gut and those oftheir homologues during Drosophila gut morphogenesis (FIG. 15). Thisconservation is of particular interest because in Drosophila the roleplayed by these genes has been clarified genetically. HH (like itsvertebrate homologue, Sonic) is expressed at the earliest stages in thegut endoderm and may be a signal to visceral mesoderm (Taylor et al.,(1993) Mech. Dev. 42:89-96). Nothing is known directly of therelationship between HH expression and activation of expression of othergenes in the Drosophila gut. However, in Drosophila imaginal discs, HHis known to activate the expression of dpp in a signaling cascade (Krauset al., (1994) Cell 75:1437-1444; Heberlein et al., (1993) Cell75:913-926; Ma et al., (1993) Cell 75:913-926; Basler et al., (1993)Cell 73 :687-702). Later in gut development, the production of dpp inthe mesoderm contributes to the regulation of the expression of homeoticgenes in both the mesoderm and the endoderm (Bienz, M. (1994) TIG10:22-26). Drosophila homeotic genes are expressed in the gut visceralmesoderm and their expression is known to determine the morphologicborders of the midgut. This involves proper induction of gene expressionin the adjacent endoderm, one of the mediators of the interaction is dpp(Bienz, M. (1994) TIG 10:22-26). If HH is required for the ultimateactivation of the homeotic genes in the Drosophila midgut, this wouldparallel the situation in the vertebrate limb bud where Sonic functionsas an upstream activator of the Hox genes (Riddle et al., (1993) Cell75:1401-1416), perhaps in a signaling cascade involving BMP-2.

[0587] The extraordinary conservation in the expression of regulatorygenes in the vertebrate and Drosophila gut strongly suggests aconservation of patterning mechanisms. Pathways established by geneticstudies in Drosophila provide direct insights into the molecular basisfor the regionalization and morphogenesis of the vertebrate gut.

EXAMPLE 9

[0588] Bacterially Expressed Hedgehog Proteins RetainMotorneuron-Inducing Activity

[0589] Various fragments of the mouse Shh gene were cloned into thepET11D vector as fusion proteins with a poly(His) leader sequence tofacilitate purification. Briefly, fusion genes encoding the mature M-Shhprotein (corresponding to Cys-25 through Ser-437 of SEQ ID No. 11) orN-terminal containing fragments, and an N-terminal exogenous leaderhaving the sequence M-G-S-S-H-H-H-H-H-H-L-V-P-R-G-S-H-M were cloned inpET11D and introduced into E. coli. The poly(His)-Shh fusion proteinswere purified using nickel chelate chromatography according to thevendor's instructions (Qiagen catalog 30210), and the poly(His) leadercleaved from the purified proteins by treatment with thrombin.

[0590] Preparations of the purified Shh proteins were added to tissueexplants (neural tube) obtained from chicken embryos and cultured in adefined media (e.g., no serum). M-Shh protein was added to finalconcentrations of between 0.5 pM to 5 nM, and differentiation of theembryonic explant tissue to motorneuron phenotype was detected byexpression of Islet-1 antigen. The bacterially produced protein wasdemonstrated to be active in the explant cultures at concentrations aslow as 5 to 50 pM. An Shh polypeptide containing all 19 kd of the aminoterminal fragment and approximately 9 kd of the carboxyl terminalfragment (see Example 6) displayed both motor neuron inducing activityand weak floor plate inducing activity, indicating that these activitieslikely reside with the N-terminal fragment.

EXAMPLE 10 Induction of Dopaminergic Neuron Phenotype with SonicHedgehog

[0591] Hamburger-Hamilton stage 8-10 chick embryos were dissected freeof the vitelline membranes and the areas opaca and pellucida. Theembryos were then incubated in Dulbecco's Modified Eagle's Mediumcontaining 0.5% dispase (Boehringer), 10 μg/ml hyaluronidase (Sigma),and 0.04% DNAse I (Sigma). The neural plate was then separated from itsunderlying mesoderm and notochord. The presumptive midbrain wasidentified and located according to its fate map (Couly and Le Douarin,1987, Developmental Biol. 120:198-214) and isolated. The ventralone-third of the mesencephalic neural plate, comprising the presumptivefloor plate and adjacent prospective dopaminergic neurons was thenremoved and discarded. The dorsal one-third was likewise dissected andremoved. The remaining intermediate region was then incubated in vitroon a 2% agarose (Sigma) containing substrate made with alpha medium(Gibco). Recombinant Shh hedgehog, both human and mouse (full lengthcDNA), was then introduced to the tissue in one of two ways: (1) Boundto nickel-agarose beads (Qiagen) via the 6-histidine tag engineered ontothe amino terminus of the protein, or (2) was incorporated in a solubleform directly into the agarose substrate. Dihydrofolate reductase wasused as the control protein for these experiments. The tissue was thenincubated at 37° C. for periods ranging from 36-48 hours. For analysis,tissue was fixed at 4° C. in 4% paraformaldehyde and stored in 50% MeOHuntil staining. Staining was done for both tyrosine hydroxylase (TH)(Boehringer), L-DOPA (Chemicon), and dopamine (DA) (Chemicon).

[0592] The data indicate that both mouse and human recombinant Shhhedgehog were active in the above described experiments. Furthermore,results indicate that addition of Shh induces both islet-1 (a motorneuron marker) and TH (a catecholaminergic neuron) as well as theaccumulation of L-DOPA in the mesencephalon, which is indicative of adopaminergic phenotype.

EXAMPLE 11 Sonic Hedgehog Induces Bone Formation

[0593] The ectopic bone formation assay was essentially done asdescribed in Sampath and Reddi, 1983, PNAS USA 80:6591-6595. The mouseShh protein was frozen and lyophilized, and the powder was enclosed inno. 5 gelatin capsule. Alternatively, 0.9-2.0 mg of collagen sponge(Collastat) was used as matrix. The Shh protein (12.5 μg) was addeddirectly to the washed sponge, the sponge lyophilized, and the spongeimplanted. The capsules or collagen sponges were implantedsubcutaneously in the abdominal thoracic area of 21- to 49-day femaleLong-Evans rats and routinely removed at 11 days. Samples were processedfor histological analysis, with 1-μm glycolmethacrylate sections stainedwith Von Kossa and acid fuschin or toluidine blue. Von Kossa stainingshows mineral (hydroxyapatite) formation. The collagen sponge by itselfwas used as a control in these experiments. The results indicate thatthe addition of mouse Shh protein induced bone formation in these rats.

[0594] All of the above-cited references and publications are herebyincorporated by reference.

Equivalents

[0595] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, numerous equivalents to thespecific polypeptides, nucleic acids, methods, assays and reagentsdescribed herein. Such equivalents are considered to be within the scopeof this invention and are covered by the following claims.

1 47 1277 base pairs nucleic acid both linear cDNA CDS 1..1275 1 ATG GTCGAA ATG CTG CTG TTG ACA AGA ATT CTC TTG GTG GGC TTC ATC 48 Met Val GluMet Leu Leu Leu Thr Arg Ile Leu Leu Val Gly Phe Ile 1 5 10 15 TGC GCTCTT TTA GTC TCC TCT GGG CTG ACT TGT GGA CCA GGC AGG GGC 96 Cys Ala LeuLeu Val Ser Ser Gly Leu Thr Cys Gly Pro Gly Arg Gly 20 25 30 ATT GGA AAAAGG AGG CAC CCC AAA AAG CTG ACC CCG TTA GCC TAT AAG 144 Ile Gly Lys ArgArg His Pro Lys Lys Leu Thr Pro Leu Ala Tyr Lys 35 40 45 CAG TTT ATT CCCAAT GTG GCA GAG AAG ACC CTA GGG GCC AGT GGA AGA 192 Gln Phe Ile Pro AsnVal Ala Glu Lys Thr Leu Gly Ala Ser Gly Arg 50 55 60 TAT GAA GGG AAG ATCACA AGA AAC TCC GAG AGA TTT AAA GAA CTA ACC 240 Tyr Glu Gly Lys Ile ThrArg Asn Ser Glu Arg Phe Lys Glu Leu Thr 65 70 75 80 CCA AAT TAC AAC CCTGAC ATT ATT TTT AAG GAT GAA GAG AAC ACG GGA 288 Pro Asn Tyr Asn Pro AspIle Ile Phe Lys Asp Glu Glu Asn Thr Gly 85 90 95 GCT GAC AGA CTG ATG ACTCAG CGC TGC AAG GAC AAG CTG AAT GCC CTG 336 Ala Asp Arg Leu Met Thr GlnArg Cys Lys Asp Lys Leu Asn Ala Leu 100 105 110 GCG ATC TCG GTG ATG AACCAG TGG CCC GGG GTG AAG CTG CGG GTG ACC 384 Ala Ile Ser Val Met Asn GlnTrp Pro Gly Val Lys Leu Arg Val Thr 115 120 125 GAG GGC TGG GAC GAG GATGGC CAT CAC TCC GAG GAA TCG CTG CAC TAC 432 Glu Gly Trp Asp Glu Asp GlyHis His Ser Glu Glu Ser Leu His Tyr 130 135 140 GAG GGT CGC GCC GTG GACATC ACC ACG TCG GAT CGG GAC CGC AGC AAG 480 Glu Gly Arg Ala Val Asp IleThr Thr Ser Asp Arg Asp Arg Ser Lys 145 150 155 160 TAC GGA ATG CTG GCCCGC CTC GCC GTC GAG GCC GGC TTC GAC TGG GTC 528 Tyr Gly Met Leu Ala ArgLeu Ala Val Glu Ala Gly Phe Asp Trp Val 165 170 175 TAC TAC GAG TCC AAGGCG CAC ATC CAC TGC TCC GTC AAA GCA GAA AAC 576 Tyr Tyr Glu Ser Lys AlaHis Ile His Cys Ser Val Lys Ala Glu Asn 180 185 190 TCA GTG GCA GCG AAATCA GGA GGC TGC TTC CCT GGC TCA GCC ACA GTG 624 Ser Val Ala Ala Lys SerGly Gly Cys Phe Pro Gly Ser Ala Thr Val 195 200 205 CAC CTG GAG CAT GGAGGC ACC AAG CTG GTG AAG GAC CTG AGC CCT GGG 672 His Leu Glu His Gly GlyThr Lys Leu Val Lys Asp Leu Ser Pro Gly 210 215 220 GAC CGC GTG CTG GCTGCT GAC GCG GAC GGC CGG CTG CTC TAC AGT GAC 720 Asp Arg Val Leu Ala AlaAsp Ala Asp Gly Arg Leu Leu Tyr Ser Asp 225 230 235 240 TTC CTC ACC TTCCTC GAC CGG ATG GAC AGC TCC CGA AAG CTC TTC TAC 768 Phe Leu Thr Phe LeuAsp Arg Met Asp Ser Ser Arg Lys Leu Phe Tyr 245 250 255 GTC ATC GAG ACGCGG CAG CCC CGG GCC CGG CTG CTA CTG ACG GCG GCC 816 Val Ile Glu Thr ArgGln Pro Arg Ala Arg Leu Leu Leu Thr Ala Ala 260 265 270 CAC CTG CTC TTTGTG GCC CCC CAG CAC AAC CAG TCG GAG GCC ACA GGG 864 His Leu Leu Phe ValAla Pro Gln His Asn Gln Ser Glu Ala Thr Gly 275 280 285 TCC ACC AGT GGCCAG GCG CTC TTC GCC AGC AAC GTG AAG CCT GGC CAA 912 Ser Thr Ser Gly GlnAla Leu Phe Ala Ser Asn Val Lys Pro Gly Gln 290 295 300 CGT GTC TAT GTGCTG GGC GAG GGC GGG CAG CAG CTG CTG CCG GCG TCT 960 Arg Val Tyr Val LeuGly Glu Gly Gly Gln Gln Leu Leu Pro Ala Ser 305 310 315 320 GTC CAC AGCGTC TCA TTG CGG GAG GAG GCG TCC GGA GCC TAC GCC CCA 1008 Val His Ser ValSer Leu Arg Glu Glu Ala Ser Gly Ala Tyr Ala Pro 325 330 335 CTC ACC GCCCAG GGC ACC ATC CTC ATC AAC CGG GTG TTG GCC TCC TGC 1056 Leu Thr Ala GlnGly Thr Ile Leu Ile Asn Arg Val Leu Ala Ser Cys 340 345 350 TAC GCC GTCATC GAG GAG CAC AGT TGG GCC CAT TGG GCC TTC GCA CCA 1104 Tyr Ala Val IleGlu Glu His Ser Trp Ala His Trp Ala Phe Ala Pro 355 360 365 TTC CGC TTGGCT CAG GGG CTG CTG GCC GCC CTC TGC CCA GAT GGG GCC 1152 Phe Arg Leu AlaGln Gly Leu Leu Ala Ala Leu Cys Pro Asp Gly Ala 370 375 380 ATC CCT ACTGCC GCC ACC ACC ACC ACT GGC ATC CAT TGG TAC TCA CGG 1200 Ile Pro Thr AlaAla Thr Thr Thr Thr Gly Ile His Trp Tyr Ser Arg 385 390 395 400 CTC CTCTAC CGC ATC GGC AGC TGG GTG CTG GAT GGT GAC GCG CTG CAT 1248 Leu Leu TyrArg Ile Gly Ser Trp Val Leu Asp Gly Asp Ala Leu His 405 410 415 CCG CTGGGC ATG GTG GCA CCG GCC AGC TG 1277 Pro Leu Gly Met Val Ala Pro Ala Ser420 425 1190 base pairs nucleic acid both linear cDNA CDS 1..1190 2 ATGGCT CTG CCG GCC AGT CTG TTG CCC CTG TGC TGC TTG GCA CTC TTG 48 Met AlaLeu Pro Ala Ser Leu Leu Pro Leu Cys Cys Leu Ala Leu Leu 1 5 10 15 GCACTA TCT GCC CAG AGC TGC GGG CCG GGC CGA GGA CCG GTT GGC CGG 96 Ala LeuSer Ala Gln Ser Cys Gly Pro Gly Arg Gly Pro Val Gly Arg 20 25 30 CGG CGTTAT GTG CGC AAG CAA CTT GTG CCT CTG CTA TAC AAG CAG TTT 144 Arg Arg TyrVal Arg Lys Gln Leu Val Pro Leu Leu Tyr Lys Gln Phe 35 40 45 GTG CCC AGTATG CCC GAG CGG ACC CTG GGC GCG AGT GGG CCA GCG GAG 192 Val Pro Ser MetPro Glu Arg Thr Leu Gly Ala Ser Gly Pro Ala Glu 50 55 60 GGG AGG GTA ACAAGG GGG TCG GAG CGC TTC CGG GAC CTC GTA CCC AAC 240 Gly Arg Val Thr ArgGly Ser Glu Arg Phe Arg Asp Leu Val Pro Asn 65 70 75 80 TAC AAC CCC GACATA ATC TTC AAG GAT GAG GAG AAC AGC GGC GCA GAC 288 Tyr Asn Pro Asp IleIle Phe Lys Asp Glu Glu Asn Ser Gly Ala Asp 85 90 95 CGC CTG ATG ACA GAGCGT TGC AAA GAG CGG GTG AAC GCT CTA GCC ATC 336 Arg Leu Met Thr Glu ArgCys Lys Glu Arg Val Asn Ala Leu Ala Ile 100 105 110 GCG GTG ATG AAC ATGTGG CCC GGA GTA CGC CTA CGT GTG ACT GAA GGC 384 Ala Val Met Asn Met TrpPro Gly Val Arg Leu Arg Val Thr Glu Gly 115 120 125 TGG GAC GAG GAC GGCCAC CAC GCA CAG GAT TCA CTC CAC TAC GAA GGC 432 Trp Asp Glu Asp Gly HisHis Ala Gln Asp Ser Leu His Tyr Glu Gly 130 135 140 CGT GCC TTG GAC ATCACC ACG TCT GAC CGT GAC CGT AAT AAG TAT GGT 480 Arg Ala Leu Asp Ile ThrThr Ser Asp Arg Asp Arg Asn Lys Tyr Gly 145 150 155 160 TTG TTG GCG CGCCTA GCT GTG GAA GCC GGA TTC GAC TGG GTC TAC TAC 528 Leu Leu Ala Arg LeuAla Val Glu Ala Gly Phe Asp Trp Val Tyr Tyr 165 170 175 GAG TCC CGC AACCAC ATC CAC GTA TCG GTC AAA GCT GAT AAC TCA CTG 576 Glu Ser Arg Asn HisIle His Val Ser Val Lys Ala Asp Asn Ser Leu 180 185 190 GCG GTC CGA GCCGGA GGC TGC TTT CCG GGA AAT GCC ACG GTG CGC TTG 624 Ala Val Arg Ala GlyGly Cys Phe Pro Gly Asn Ala Thr Val Arg Leu 195 200 205 CGG AGC GGC GAACGG AAG GGG CTG AGG GAA CTA CAT CGT GGT GAC TGG 672 Arg Ser Gly Glu ArgLys Gly Leu Arg Glu Leu His Arg Gly Asp Trp 210 215 220 GTA CTG GCC GCTGAT GCA GCG GGC CGA GTG GTA CCC ACG CCA GTG CTG 720 Val Leu Ala Ala AspAla Ala Gly Arg Val Val Pro Thr Pro Val Leu 225 230 235 240 CTC TTC CTGGAC CGG GAT CTG CAG CGC CGC GCC TCG TTC GTG GCT GTG 768 Leu Phe Leu AspArg Asp Leu Gln Arg Arg Ala Ser Phe Val Ala Val 245 250 255 GAG ACC GAGCGG CCT CCG CGC AAA CTG TTG CTC ACA CCC TGG CAT CTG 816 Glu Thr Glu ArgPro Pro Arg Lys Leu Leu Leu Thr Pro Trp His Leu 260 265 270 GTG TTC GCTGCT CGC GGG CCA GCG CCT GCT CCA GGT GAC TTT GCA CCG 864 Val Phe Ala AlaArg Gly Pro Ala Pro Ala Pro Gly Asp Phe Ala Pro 275 280 285 GTG TTC GCGCGC CGC TTA CGT GCT GGC GAC TCG GTG CTG GCT CCC GGC 912 Val Phe Ala ArgArg Leu Arg Ala Gly Asp Ser Val Leu Ala Pro Gly 290 295 300 GGG GAC GCGCTC CAG CCG GCG CGC GTA GCC CGC GTG GCG CGC GAG GAA 960 Gly Asp Ala LeuGln Pro Ala Arg Val Ala Arg Val Ala Arg Glu Glu 305 310 315 320 GCC GTGGGC GTG TTC GCA CCG CTC ACT GCG CAC GGG ACG CTG CTG GTC 1008 Ala Val GlyVal Phe Ala Pro Leu Thr Ala His Gly Thr Leu Leu Val 325 330 335 AAC GACGTC CTC GCC TCC TGC TAC GCG GTT CTA GAG AGT CAC CAG TGG 1056 Asn Asp ValLeu Ala Ser Cys Tyr Ala Val Leu Glu Ser His Gln Trp 340 345 350 GCC CACCGC GCC TTC GCC CCT TTG CGG CTG CTG CAC GCG CTC GGG GCT 1104 Ala His ArgAla Phe Ala Pro Leu Arg Leu Leu His Ala Leu Gly Ala 355 360 365 CTG CTCCCT GGG GGT GCA GTC CAG CCG ACT GGC ATG CAT TGG TAC TCT 1152 Leu Leu ProGly Gly Ala Val Gln Pro Thr Gly Met His Trp Tyr Ser 370 375 380 CGC CTCCTT TAC CGC TTG GCC GAG GAG TTA ATG GGC TG 1190 Arg Leu Leu Tyr Arg LeuAla Glu Glu Leu Met Gly 385 390 395 1281 base pairs nucleic acid bothlinear cDNA CDS 1..1233 3 ATG TCT CCC GCC TGG CTC CGG CCC CGA CTG CGGTTC TGT CTG TTC CTG 48 Met Ser Pro Ala Trp Leu Arg Pro Arg Leu Arg PheCys Leu Phe Leu 1 5 10 15 CTG CTG CTG CTT CTG GTG CCG GCG GCG CGG GGCTGC GGG CCG GGC CGG 96 Leu Leu Leu Leu Leu Val Pro Ala Ala Arg Gly CysGly Pro Gly Arg 20 25 30 GTG GTG GGC AGC CGC CGG AGG CCG CCT CGC AAG CTCGTG CCT CTT GCC 144 Val Val Gly Ser Arg Arg Arg Pro Pro Arg Lys Leu ValPro Leu Ala 35 40 45 TAC AAG CAG TTC AGC CCC AAC GTG CCG GAG AAG ACC CTGGGC GCC AGC 192 Tyr Lys Gln Phe Ser Pro Asn Val Pro Glu Lys Thr Leu GlyAla Ser 50 55 60 GGG CGC TAC GAA GGC AAG ATC GCG CGC AGC TCT GAG CGC TTCAAA GAG 240 Gly Arg Tyr Glu Gly Lys Ile Ala Arg Ser Ser Glu Arg Phe LysGlu 65 70 75 80 CTC ACC CCC AAC TAC AAT CCC GAC ATC ATC TTC AAG GAC GAGGAG AAC 288 Leu Thr Pro Asn Tyr Asn Pro Asp Ile Ile Phe Lys Asp Glu GluAsn 85 90 95 ACG GGT GCC GAC CGC CTC ATG ACC CAG CGC TGC AAG GAC CGT CTGAAC 336 Thr Gly Ala Asp Arg Leu Met Thr Gln Arg Cys Lys Asp Arg Leu Asn100 105 110 TCA CTG GCC ATC TCT GTC ATG AAC CAG TGG CCT GGT GTG AAA CTGCGG 384 Ser Leu Ala Ile Ser Val Met Asn Gln Trp Pro Gly Val Lys Leu Arg115 120 125 GTG ACC GAA GGC CGG GAT GAA GAT GGC CAT CAC TCA GAG GAG TCTTTA 432 Val Thr Glu Gly Arg Asp Glu Asp Gly His His Ser Glu Glu Ser Leu130 135 140 CAC TAT GAG GGC CGC GCG GTG GAT ATC ACC ACC TCA GAC CGT GACCGA 480 His Tyr Glu Gly Arg Ala Val Asp Ile Thr Thr Ser Asp Arg Asp Arg145 150 155 160 AAT AAG TAT GGA CTG CTG GCG CGC TTA GCA GTG GAG GCC GGCTTC GAC 528 Asn Lys Tyr Gly Leu Leu Ala Arg Leu Ala Val Glu Ala Gly PheAsp 165 170 175 TGG GTG TAT TAC GAG TCC AAG GCC CAC GTG CAT TGC TCT GTCAAG TCT 576 Trp Val Tyr Tyr Glu Ser Lys Ala His Val His Cys Ser Val LysSer 180 185 190 GAG CAT TCG GCC GCT GCC AAG ACA GGT GGC TGC TTT CCT GCCGGA GCC 624 Glu His Ser Ala Ala Ala Lys Thr Gly Gly Cys Phe Pro Ala GlyAla 195 200 205 CAG GTG CGC CTA GAG AAC GGG GAG CGT GTG GCC CTG TCA GCTGTA AAG 672 Gln Val Arg Leu Glu Asn Gly Glu Arg Val Ala Leu Ser Ala ValLys 210 215 220 CCA GGA GAC CGG GTG CTG GCC ATG GGG GAG GAT GGG ACC CCCACC TTC 720 Pro Gly Asp Arg Val Leu Ala Met Gly Glu Asp Gly Thr Pro ThrPhe 225 230 235 240 AGT GAT GTG CTT ATT TTC CTG GAC CGC GAG CCA AAC CGGCTG AGA GCT 768 Ser Asp Val Leu Ile Phe Leu Asp Arg Glu Pro Asn Arg LeuArg Ala 245 250 255 TTC CAG GTC ATC GAG ACT CAG GAT CCT CCG CGT CGG CTGGCG CTC ACG 816 Phe Gln Val Ile Glu Thr Gln Asp Pro Pro Arg Arg Leu AlaLeu Thr 260 265 270 CCT GCC CAC CTG CTC TTC ATT GCG GAC AAT CAT ACA GAACCA GCA GCC 864 Pro Ala His Leu Leu Phe Ile Ala Asp Asn His Thr Glu ProAla Ala 275 280 285 CAC TTC CGG GCC ACA TTT GCC AGC CAT GTG CAA CCA GGCCAA TAT GTG 912 His Phe Arg Ala Thr Phe Ala Ser His Val Gln Pro Gly GlnTyr Val 290 295 300 CTG GTA TCA GGG GTA CCA GGC CTC CAG CCT GCT CGG GTGGCA GCT GTC 960 Leu Val Ser Gly Val Pro Gly Leu Gln Pro Ala Arg Val AlaAla Val 305 310 315 320 TCC ACC CAC GTG GCC CTT GGG TCC TAT GCT CCT CTCACA AGG CAT GGG 1008 Ser Thr His Val Ala Leu Gly Ser Tyr Ala Pro Leu ThrArg His Gly 325 330 335 ACA CTT GTG GTG GAG GAT GTG GTG GCC TCC TGC TTTGCA GCT GTG GCT 1056 Thr Leu Val Val Glu Asp Val Val Ala Ser Cys Phe AlaAla Val Ala 340 345 350 GAC CAC CAT CTG GCT CAG TTG GCC TTC TGG CCC CTGCGA CTG TTT CCC 1104 Asp His His Leu Ala Gln Leu Ala Phe Trp Pro Leu ArgLeu Phe Pro 355 360 365 AGT TTG GCA TGG GGC AGC TGG ACC CCA AGT GAG GGTGTT CAC TCC TAC 1152 Ser Leu Ala Trp Gly Ser Trp Thr Pro Ser Glu Gly ValHis Ser Tyr 370 375 380 CCT CAG ATG CTC TAC CGC CTG GGG CGT CTC TTG CTAGAA GAG AGC ACC 1200 Pro Gln Met Leu Tyr Arg Leu Gly Arg Leu Leu Leu GluGlu Ser Thr 385 390 395 400 TTC CAT CCA CTG GGC ATG TCT GGG GCA GGA AGCTGAAGGGACT CTAACCACTG 1253 Phe His Pro Leu Gly Met Ser Gly Ala Gly Ser405 410 CCCTCCTGGA ACTGCTGTGC GTGGATCC 1281 1313 base pairs nucleic acidboth linear cDNA CDS 1..1313 4 ATG CTG CTG CTG CTG GCC AGA TGT TTT CTGGTG ATC CTT GCT TCC TCG 48 Met Leu Leu Leu Leu Ala Arg Cys Phe Leu ValIle Leu Ala Ser Ser 1 5 10 15 CTG CTG GTG TGC CCC GGG CTG GCC TGT GGGCCC GGC AGG GGG TTT GGA 96 Leu Leu Val Cys Pro Gly Leu Ala Cys Gly ProGly Arg Gly Phe Gly 20 25 30 AAG AGG CGG CAC CCC AAA AAG CTG ACC CCT TTAGCC TAC AAG CAG TTT 144 Lys Arg Arg His Pro Lys Lys Leu Thr Pro Leu AlaTyr Lys Gln Phe 35 40 45 ATT CCC AAC GTA GCC GAG AAG ACC CTA GGG GCC AGCGGC AGA TAT GAA 192 Ile Pro Asn Val Ala Glu Lys Thr Leu Gly Ala Ser GlyArg Tyr Glu 50 55 60 GGG AAG ATC ACA AGA AAC TCC GAA CGA TTT AAG GAA CTCACC CCC AAT 240 Gly Lys Ile Thr Arg Asn Ser Glu Arg Phe Lys Glu Leu ThrPro Asn 65 70 75 80 TAC AAC CCC GAC ATC ATA TTT AAG GAT GAG GAA AAC ACGGGA GCA GAC 288 Tyr Asn Pro Asp Ile Ile Phe Lys Asp Glu Glu Asn Thr GlyAla Asp 85 90 95 CGG CTG ATG ACT CAG AGG TGC AAA GAC AAG TTA AAT GCC TTGGCC ATC 336 Arg Leu Met Thr Gln Arg Cys Lys Asp Lys Leu Asn Ala Leu AlaIle 100 105 110 TCT GTG ATG AAC CAG TGG CCT GGA GTG AGG CTG CGA GTG ACCGAG GGC 384 Ser Val Met Asn Gln Trp Pro Gly Val Arg Leu Arg Val Thr GluGly 115 120 125 TGG GAT GAG GAC GGC CAT CAT TCA GAG GAG TCT CTA CAC TATGAG GGT 432 Trp Asp Glu Asp Gly His His Ser Glu Glu Ser Leu His Tyr GluGly 130 135 140 CGA GCA GTG GAC ATC ACC ACG TCC GAC CGG GAC CGC AGC AAGTAC GGC 480 Arg Ala Val Asp Ile Thr Thr Ser Asp Arg Asp Arg Ser Lys TyrGly 145 150 155 160 ATG CTG GCT CGC CTG GCT GTG GAA GCA GGT TTC GAC TGGGTC TAC TAT 528 Met Leu Ala Arg Leu Ala Val Glu Ala Gly Phe Asp Trp ValTyr Tyr 165 170 175 GAA TCC AAA GCT CAC ATC CAC TGT TCT GTG AAA GCA GAGAAC TCC GTG 576 Glu Ser Lys Ala His Ile His Cys Ser Val Lys Ala Glu AsnSer Val 180 185 190 GCG GCC AAA TCC GGC GGC TGT TTC CCG GGA TCC GCC ACCGTG CAC CTG 624 Ala Ala Lys Ser Gly Gly Cys Phe Pro Gly Ser Ala Thr ValHis Leu 195 200 205 GAG CAG GGC GGC ACC AAG CTG GTG AAG GAC TTA CGT CCCGGA GAC CGC 672 Glu Gln Gly Gly Thr Lys Leu Val Lys Asp Leu Arg Pro GlyAsp Arg 210 215 220 GTG CTG GCG GCT GAC GAC CAG GGC CGG CTG CTG TAC AGCGAC TTC CTC 720 Val Leu Ala Ala Asp Asp Gln Gly Arg Leu Leu Tyr Ser AspPhe Leu 225 230 235 240 ACC TTC CTG GAC CGC GAC GAA GGC GCC AAG AAG GTCTTC TAC GTG ATC 768 Thr Phe Leu Asp Arg Asp Glu Gly Ala Lys Lys Val PheTyr Val Ile 245 250 255 GAG ACG CTG GAG CCG CGC GAG CGC CTG CTG CTC ACCGCC GCG CAC CTG 816 Glu Thr Leu Glu Pro Arg Glu Arg Leu Leu Leu Thr AlaAla His Leu 260 265 270 CTC TTC GTG GCG CCG CAC AAC GAC TCG GGG CCC ACGCCC GGG CCA AGC 864 Leu Phe Val Ala Pro His Asn Asp Ser Gly Pro Thr ProGly Pro Ser 275 280 285 GCG CTC TTT GCC AGC CGC GTG CGC CCC GGG CAG CGCGTG TAC GTG GTG 912 Ala Leu Phe Ala Ser Arg Val Arg Pro Gly Gln Arg ValTyr Val Val 290 295 300 GCT GAA CGC GGC GGG GAC CGC CGG CTG CTG CCC GCCGCG GTG CAC AGC 960 Ala Glu Arg Gly Gly Asp Arg Arg Leu Leu Pro Ala AlaVal His Ser 305 310 315 320 GTG ACG CTG CGA GAG GAG GAG GCG GGC GCG TACGCG CCG CTC ACG GCG 1008 Val Thr Leu Arg Glu Glu Glu Ala Gly Ala Tyr AlaPro Leu Thr Ala 325 330 335 CAC GGC ACC ATT CTC ATC AAC CGG GTG CTC GCCTCG TGC TAC GCT GTC 1056 His Gly Thr Ile Leu Ile Asn Arg Val Leu Ala SerCys Tyr Ala Val 340 345 350 ATC GAG GAG CAC AGC TGG GCA CAC CGG GCC TTCGCG CCT TTC CGC CTG 1104 Ile Glu Glu His Ser Trp Ala His Arg Ala Phe AlaPro Phe Arg Leu 355 360 365 GCG CAC GCG CTG CTG GCC GCG CTG GCA CCC GCCCGC ACG GAC GGC GGG 1152 Ala His Ala Leu Leu Ala Ala Leu Ala Pro Ala ArgThr Asp Gly Gly 370 375 380 GGC GGG GGC AGC ATC CCT GCA GCG CAA TCT GCAACG GAA GCG AGG GGC 1200 Gly Gly Gly Ser Ile Pro Ala Ala Gln Ser Ala ThrGlu Ala Arg Gly 385 390 395 400 GCG GAG CCG ACT GCG GGC ATC CAC TGG TACTCG CAG CTG CTC TAC CAC 1248 Ala Glu Pro Thr Ala Gly Ile His Trp Tyr SerGln Leu Leu Tyr His 405 410 415 ATT GGC ACC TGG CTG TTG GAC AGC GAG ACCATG CAT CCC TTG GGA ATG 1296 Ile Gly Thr Trp Leu Leu Asp Ser Glu Thr MetHis Pro Leu Gly Met 420 425 430 GCG GTC AAG TCC AGC TG 1313 Ala Val LysSer Ser 435 1256 base pairs nucleic acid both linear cDNA CDS 1..1256 5ATG CGG CTT TTG ACG AGA GTG CTG CTG GTG TCT CTT CTC ACT CTG TCC 48 MetArg Leu Leu Thr Arg Val Leu Leu Val Ser Leu Leu Thr Leu Ser 1 5 10 15TTG GTG GTG TCC GGA CTG GCC TGC GGT CCT GGC AGA GGC TAC GGC AGA 96 LeuVal Val Ser Gly Leu Ala Cys Gly Pro Gly Arg Gly Tyr Gly Arg 20 25 30 AGAAGA CAT CCG AAG AAG CTG ACA CCT CTC GCC TAC AAG CAG TTC ATA 144 Arg ArgHis Pro Lys Lys Leu Thr Pro Leu Ala Tyr Lys Gln Phe Ile 35 40 45 CCT AATGTC GCG GAG AAG ACC TTA GGG GCC AGC GGC AGA TAC GAG GGC 192 Pro Asn ValAla Glu Lys Thr Leu Gly Ala Ser Gly Arg Tyr Glu Gly 50 55 60 AAG ATA ACGCGC AAT TCG GAG AGA TTT AAA GAA CTT ACT CCA AAT TAC 240 Lys Ile Thr ArgAsn Ser Glu Arg Phe Lys Glu Leu Thr Pro Asn Tyr 65 70 75 80 AAT CCC GACATT ATC TTT AAG GAT GAG GAG AAC ACG GGA GCG GAC AGG 288 Asn Pro Asp IleIle Phe Lys Asp Glu Glu Asn Thr Gly Ala Asp Arg 85 90 95 CTC ATG ACA CAGAGA TGC AAA GAC AAG CTG AAC TCG CTG GCC ATC TCT 336 Leu Met Thr Gln ArgCys Lys Asp Lys Leu Asn Ser Leu Ala Ile Ser 100 105 110 GTA ATG AAC CACTGG CCA GGG GTT AAG CTG CGT GTG ACA GAG GGC TGG 384 Val Met Asn His TrpPro Gly Val Lys Leu Arg Val Thr Glu Gly Trp 115 120 125 GAT GAG GAC GGTCAC CAT TTT GAA GAA TCA CTC CAC TAC GAG GGA AGA 432 Asp Glu Asp Gly HisHis Phe Glu Glu Ser Leu His Tyr Glu Gly Arg 130 135 140 GCT GTT GAT ATTACC ACC TCT GAC CGA GAC AAG AGC AAA TAC GGG ACA 480 Ala Val Asp Ile ThrThr Ser Asp Arg Asp Lys Ser Lys Tyr Gly Thr 145 150 155 160 CTG TCT CGCCTA GCT GTG GAG GCT GGA TTT GAC TGG GTC TAT TAC GAG 528 Leu Ser Arg LeuAla Val Glu Ala Gly Phe Asp Trp Val Tyr Tyr Glu 165 170 175 TCC AAA GCCCAC ATT CAT TGC TCT GTC AAA GCA GAA AAT TCG GTT GCT 576 Ser Lys Ala HisIle His Cys Ser Val Lys Ala Glu Asn Ser Val Ala 180 185 190 GCG AAA TCTGGG GGC TGT TTC CCA GGT TCG GCT CTG GTC TCG CTC CAG 624 Ala Lys Ser GlyGly Cys Phe Pro Gly Ser Ala Leu Val Ser Leu Gln 195 200 205 GAC GGA GGACAG AAG GCC GTG AAG GAC CTG AAC CCC GGA GAC AAG GTG 672 Asp Gly Gly GlnLys Ala Val Lys Asp Leu Asn Pro Gly Asp Lys Val 210 215 220 CTG GCG GCAGAC AGC GCG GGA AAC CTG GTG TTC AGC GAC TTC ATC ATG 720 Leu Ala Ala AspSer Ala Gly Asn Leu Val Phe Ser Asp Phe Ile Met 225 230 235 240 TTC ACAGAC CGA GAC TCC ACG ACG CGA CGT GTG TTT TAC GTC ATA GAA 768 Phe Thr AspArg Asp Ser Thr Thr Arg Arg Val Phe Tyr Val Ile Glu 245 250 255 ACG CAAGAA CCC GTT GAA AAG ATC ACC CTC ACC GCC GCT CAC CTC CTT 816 Thr Gln GluPro Val Glu Lys Ile Thr Leu Thr Ala Ala His Leu Leu 260 265 270 TTT GTCCTC GAC AAC TCA ACG GAA GAT CTC CAC ACC ATG ACC GCC GCG 864 Phe Val LeuAsp Asn Ser Thr Glu Asp Leu His Thr Met Thr Ala Ala 275 280 285 TAT GCCAGC AGT GTC AGA GCC GGA CAA AAG GTG ATG GTT GTT GAT GAT 912 Tyr Ala SerSer Val Arg Ala Gly Gln Lys Val Met Val Val Asp Asp 290 295 300 AGC GGTCAG CTT AAA TCT GTC ATC GTG CAG CGG ATA TAC ACG GAG GAG 960 Ser Gly GlnLeu Lys Ser Val Ile Val Gln Arg Ile Tyr Thr Glu Glu 305 310 315 320 CAGCGG GGC TCG TTC GCA CCA GTG ACT GCA CAT GGG ACC ATT GTG GTC 1008 Gln ArgGly Ser Phe Ala Pro Val Thr Ala His Gly Thr Ile Val Val 325 330 335 GACAGA ATA CTG GCG TCC TGT TAC GCC GTA ATA GAG GAC CAG GGG CTT 1056 Asp ArgIle Leu Ala Ser Cys Tyr Ala Val Ile Glu Asp Gln Gly Leu 340 345 350 GCGCAT TTG GCC TTC GCG CCC GCC AGG CTC TAT TAT TAC GTG TCA TCA 1104 Ala HisLeu Ala Phe Ala Pro Ala Arg Leu Tyr Tyr Tyr Val Ser Ser 355 360 365 TTCCTG TCC CCC AAA ACT CCA GCA GTC GGT CCA ATG CGA CTT TAC AAC 1152 Phe LeuSer Pro Lys Thr Pro Ala Val Gly Pro Met Arg Leu Tyr Asn 370 375 380 AGGAGG GGG TCC ACT GGT ACT CCA GGC TCC TGT CAT CAA ATG GGA ACG 1200 Arg ArgGly Ser Thr Gly Thr Pro Gly Ser Cys His Gln Met Gly Thr 385 390 395 400TGG CTT TTG GAC AGC AAC ATG CTT CAT CCT TTG GGG ATG TCA GTA AAC 1248 TrpLeu Leu Asp Ser Asn Met Leu His Pro Leu Gly Met Ser Val Asn 405 410 415TCA AGC TG 1256 Ser Ser 1425 base pairs nucleic acid single linear cDNACDS 1..1425 6 ATG CTG CTG CTG GCG AGA TGT CTG CTG CTA GTC CTC GTC TCCTCG CTG 48 Met Leu Leu Leu Ala Arg Cys Leu Leu Leu Val Leu Val Ser SerLeu 1 5 10 15 CTG GTA TGC TCG GGA CTG GCG TGC GGA CCG GGC AGG GGG TTCGGG AAG 96 Leu Val Cys Ser Gly Leu Ala Cys Gly Pro Gly Arg Gly Phe GlyLys 20 25 30 AGG AGG CAC CCC AAA AAG CTG ACC CCT TTA GCC TAC AAG CAG TTTATC 144 Arg Arg His Pro Lys Lys Leu Thr Pro Leu Ala Tyr Lys Gln Phe Ile35 40 45 CCC AAT GTG GCC GAG AAG ACC CTA GGC GCC AGC GGA AGG TAT GAA GGG192 Pro Asn Val Ala Glu Lys Thr Leu Gly Ala Ser Gly Arg Tyr Glu Gly 5055 60 AAG ATC TCC AGA AAC TCC GAG CGA TTT AAG GAA CTC ACC CCC AAT TAC240 Lys Ile Ser Arg Asn Ser Glu Arg Phe Lys Glu Leu Thr Pro Asn Tyr 6570 75 80 AAC CCC GAC ATC ATA TTT AAG GAT GAA GAA AAC ACC GGA GCG GAC AGG288 Asn Pro Asp Ile Ile Phe Lys Asp Glu Glu Asn Thr Gly Ala Asp Arg 8590 95 CTG ATG ACT CAG AGG TGT AAG GAC AAG TTG AAC GCT TTG GCC ATC TCG336 Leu Met Thr Gln Arg Cys Lys Asp Lys Leu Asn Ala Leu Ala Ile Ser 100105 110 GTG ATG AAC CAG TGG CCA GGA GTG AAA CTG CGG GTG ACC GAG GGC TGG384 Val Met Asn Gln Trp Pro Gly Val Lys Leu Arg Val Thr Glu Gly Trp 115120 125 GAC GAA GAT GGC CAC CAC TCA GAG GAG TCT CTG CAC TAC GAG GGC CGC432 Asp Glu Asp Gly His His Ser Glu Glu Ser Leu His Tyr Glu Gly Arg 130135 140 GCA GTG GAC ATC ACC ACG TCT GAC CGC GAC CGC AGC AAG TAC GGC ATG480 Ala Val Asp Ile Thr Thr Ser Asp Arg Asp Arg Ser Lys Tyr Gly Met 145150 155 160 CTG GCC CGC CTG GCG GTG GAG GCC GGC TTC GAC TGG GTG TAC TACGAG 528 Leu Ala Arg Leu Ala Val Glu Ala Gly Phe Asp Trp Val Tyr Tyr Glu165 170 175 TCC AAG GCA CAT ATC CAC TGC TCG GTG AAA GCA GAG AAC TCG GTGGCG 576 Ser Lys Ala His Ile His Cys Ser Val Lys Ala Glu Asn Ser Val Ala180 185 190 GCC AAA TCG GGA GGC TGC TTC CCG GGC TCG GCC ACG GTG CAC CTGGAG 624 Ala Lys Ser Gly Gly Cys Phe Pro Gly Ser Ala Thr Val His Leu Glu195 200 205 CAG GGC GGC ACC AAG CTG GTG AAG GAC CTG AGC CCC GGG GAC CGCGTG 672 Gln Gly Gly Thr Lys Leu Val Lys Asp Leu Ser Pro Gly Asp Arg Val210 215 220 CTG GCG GCG GAC GAC CAG GGC CGG CTG CTC TAC AGC GAC TTC CTCACT 720 Leu Ala Ala Asp Asp Gln Gly Arg Leu Leu Tyr Ser Asp Phe Leu Thr225 230 235 240 TTC CTG GAC CGC GAC GAC GGC GCC AAG AAG GTC TTC TAC GTGATC GAG 768 Phe Leu Asp Arg Asp Asp Gly Ala Lys Lys Val Phe Tyr Val IleGlu 245 250 255 ACG CGG GAG CCG CGC GAG CGC CTG CTG CTC ACC GCC GCG CACCTG CTC 816 Thr Arg Glu Pro Arg Glu Arg Leu Leu Leu Thr Ala Ala His LeuLeu 260 265 270 TTT GTG GCG CCG CAC AAC GAC TCG GCC ACC GGG GAG CCC GAGGCG TCC 864 Phe Val Ala Pro His Asn Asp Ser Ala Thr Gly Glu Pro Glu AlaSer 275 280 285 TCG GGC TCG GGG CCG CCT TCC GGG GGC GCA CTG GGG CCT CGGGCG CTG 912 Ser Gly Ser Gly Pro Pro Ser Gly Gly Ala Leu Gly Pro Arg AlaLeu 290 295 300 TTC GCC AGC CGC GTG CGC CCG GGC CAG CGC GTG TAC GTG GTGGCC GAG 960 Phe Ala Ser Arg Val Arg Pro Gly Gln Arg Val Tyr Val Val AlaGlu 305 310 315 320 CGT GAC GGG GAC CGC CGG CTC CTG CCC GCC GCT GTG CACAGC GTG ACC 1008 Arg Asp Gly Asp Arg Arg Leu Leu Pro Ala Ala Val His SerVal Thr 325 330 335 CTA AGC GAG GAG GCC GCG GGC GCC TAC GCG CCG CTC ACGGCC CAG GGC 1056 Leu Ser Glu Glu Ala Ala Gly Ala Tyr Ala Pro Leu Thr AlaGln Gly 340 345 350 ACC ATT CTC ATC AAC CGG GTG CTG GCC TCG TGC TAC GCGGTC ATC GAG 1104 Thr Ile Leu Ile Asn Arg Val Leu Ala Ser Cys Tyr Ala ValIle Glu 355 360 365 GAG CAC AGC TGG GCG CAC CGG GCC TTC GCG CCC TTC CGCCTG GCG CAC 1152 Glu His Ser Trp Ala His Arg Ala Phe Ala Pro Phe Arg LeuAla His 370 375 380 GCG CTC CTG GCT GCA CTG GCG CCC GCG CGC ACG GAC CGCGGC GGG GAC 1200 Ala Leu Leu Ala Ala Leu Ala Pro Ala Arg Thr Asp Arg GlyGly Asp 385 390 395 400 AGC GGC GGC GGG GAC CGC GGG GGC GGC GGC GGC AGAGTA GCC CTA ACC 1248 Ser Gly Gly Gly Asp Arg Gly Gly Gly Gly Gly Arg ValAla Leu Thr 405 410 415 GCT CCA GGT GCT GCC GAC GCT CCG GGT GCG GGG GCCACC GCG GGC ATC 1296 Ala Pro Gly Ala Ala Asp Ala Pro Gly Ala Gly Ala ThrAla Gly Ile 420 425 430 CAC TGG TAC TCG CAG CTG CTC TAC CAA ATA GGC ACCTGG CTC CTG GAC 1344 His Trp Tyr Ser Gln Leu Leu Tyr Gln Ile Gly Thr TrpLeu Leu Asp 435 440 445 AGC GAG GCC CTG CAC CCG CTG GGC ATG GCG GTC AAGTCC AGC NNN AGC 1392 Ser Glu Ala Leu His Pro Leu Gly Met Ala Val Lys SerSer Xaa Ser 450 455 460 CGG GGG GCC GGG GGA GGG GCG CGG GAG GGG GCC 1425Arg Gly Ala Gly Gly Gly Ala Arg Glu Gly Ala 465 470 475 939 base pairsnucleic acid single linear cDNA CDS 1..939 7 CGG CGC CTC ATG ACC CAG CGCTGC AAG GAC CGC CTG AAC TCG CTG GCT 48 Arg Arg Leu Met Thr Gln Arg CysLys Asp Arg Leu Asn Ser Leu Ala 1 5 10 15 ATC TCG GTG ATG AAC CAG TGGCCC GGT GTG AAG CTG CGG GTG ACC GAG 96 Ile Ser Val Met Asn Gln Trp ProGly Val Lys Leu Arg Val Thr Glu 20 25 30 GGC TGG GAC GAG GAC GGC CAC CACTCA GAG GAG TCC CTG CAT TAT GAG 144 Gly Trp Asp Glu Asp Gly His His SerGlu Glu Ser Leu His Tyr Glu 35 40 45 GGC CGC GCG GTG GAC ATC ACC ACA TCAGAC CGC GAC CGC AAT AAG TAT 192 Gly Arg Ala Val Asp Ile Thr Thr Ser AspArg Asp Arg Asn Lys Tyr 50 55 60 GGA CTG CTG GCG CGC TTG GCA GTG GAG GCCGGC TTT GAC TGG GTG TAT 240 Gly Leu Leu Ala Arg Leu Ala Val Glu Ala GlyPhe Asp Trp Val Tyr 65 70 75 80 TAC GAG TCA AAG GCC CAC GTG CAT TGC TCCGTC AAG TCC GAG CAC TCG 288 Tyr Glu Ser Lys Ala His Val His Cys Ser ValLys Ser Glu His Ser 85 90 95 GCC GCA GCC AAG ACG GGC GGC TGC TTC CCT GCCGGA GCC CAG GTA CGC 336 Ala Ala Ala Lys Thr Gly Gly Cys Phe Pro Ala GlyAla Gln Val Arg 100 105 110 CTG GAG AGT GGG GCG CGT GTG GCC TTG TCA GCCGTG AGG CCG GGA GAC 384 Leu Glu Ser Gly Ala Arg Val Ala Leu Ser Ala ValArg Pro Gly Asp 115 120 125 CGT GTG CTG GCC ATG GGG GAG GAT GGG AGC CCCACC TTC AGC GAT GTG 432 Arg Val Leu Ala Met Gly Glu Asp Gly Ser Pro ThrPhe Ser Asp Val 130 135 140 CTC ATT TTC CTG GAC CGC GAG CCC CAC AGG CTGAGA GCC TTC CAG GTC 480 Leu Ile Phe Leu Asp Arg Glu Pro His Arg Leu ArgAla Phe Gln Val 145 150 155 160 ATC GAG ACT CAG GAC CCC CCA CGC CGC CTGGCA CTC ACA CCC GCT CAC 528 Ile Glu Thr Gln Asp Pro Pro Arg Arg Leu AlaLeu Thr Pro Ala His 165 170 175 CTG CTC TTT ACG GCT GAC AAT CAC ACG GAGCCG GCA GCC CGC TTC CGG 576 Leu Leu Phe Thr Ala Asp Asn His Thr Glu ProAla Ala Arg Phe Arg 180 185 190 GCC ACA TTT GCC AGC CAC GTG CAG CCT GGCCAG TAC GTG CTG GTG GCT 624 Ala Thr Phe Ala Ser His Val Gln Pro Gly GlnTyr Val Leu Val Ala 195 200 205 GGG GTG CCA GGC CTG CAG CCT GCC CGC GTGGCA GCT GTC TCT ACA CAC 672 Gly Val Pro Gly Leu Gln Pro Ala Arg Val AlaAla Val Ser Thr His 210 215 220 GTG GCC CTC GGG GCC TAC GCC CCG CTC ACAAAG CAT GGG ACA CTG GTG 720 Val Ala Leu Gly Ala Tyr Ala Pro Leu Thr LysHis Gly Thr Leu Val 225 230 235 240 GTG GAG GAT GTG GTG GCA TCC TGC TTCGCG GCC GTG GCT GAC CAC CAC 768 Val Glu Asp Val Val Ala Ser Cys Phe AlaAla Val Ala Asp His His 245 250 255 CTG GCT CAG TTG GCC TTC TGG CCC CTGAGA CTC TTT CAC AGC TTG GCA 816 Leu Ala Gln Leu Ala Phe Trp Pro Leu ArgLeu Phe His Ser Leu Ala 260 265 270 TGG GGC AGC TGG ACC CCG GGG GAG GGTGTG CAT TGG TAC CCC CAG CTG 864 Trp Gly Ser Trp Thr Pro Gly Glu Gly ValHis Trp Tyr Pro Gln Leu 275 280 285 CTC TAC CGC CTG GGG CGT CTC CTG CTAGAA GAG GGC AGC TTC CAC CCA 912 Leu Tyr Arg Leu Gly Arg Leu Leu Leu GluGlu Gly Ser Phe His Pro 290 295 300 CTG GGC ATG TCC GGG GCA GGG AGC TGA939 Leu Gly Met Ser Gly Ala Gly Ser Xaa 305 310 425 amino acids aminoacid linear protein 8 Met Val Glu Met Leu Leu Leu Thr Arg Ile Leu LeuVal Gly Phe Ile 1 5 10 15 Cys Ala Leu Leu Val Ser Ser Gly Leu Thr CysGly Pro Gly Arg Gly 20 25 30 Ile Gly Lys Arg Arg His Pro Lys Lys Leu ThrPro Leu Ala Tyr Lys 35 40 45 Gln Phe Ile Pro Asn Val Ala Glu Lys Thr LeuGly Ala Ser Gly Arg 50 55 60 Tyr Glu Gly Lys Ile Thr Arg Asn Ser Glu ArgPhe Lys Glu Leu Thr 65 70 75 80 Pro Asn Tyr Asn Pro Asp Ile Ile Phe LysAsp Glu Glu Asn Thr Gly 85 90 95 Ala Asp Arg Leu Met Thr Gln Arg Cys LysAsp Lys Leu Asn Ala Leu 100 105 110 Ala Ile Ser Val Met Asn Gln Trp ProGly Val Lys Leu Arg Val Thr 115 120 125 Glu Gly Trp Asp Glu Asp Gly HisHis Ser Glu Glu Ser Leu His Tyr 130 135 140 Glu Gly Arg Ala Val Asp IleThr Thr Ser Asp Arg Asp Arg Ser Lys 145 150 155 160 Tyr Gly Met Leu AlaArg Leu Ala Val Glu Ala Gly Phe Asp Trp Val 165 170 175 Tyr Tyr Glu SerLys Ala His Ile His Cys Ser Val Lys Ala Glu Asn 180 185 190 Ser Val AlaAla Lys Ser Gly Gly Cys Phe Pro Gly Ser Ala Thr Val 195 200 205 His LeuGlu His Gly Gly Thr Lys Leu Val Lys Asp Leu Ser Pro Gly 210 215 220 AspArg Val Leu Ala Ala Asp Ala Asp Gly Arg Leu Leu Tyr Ser Asp 225 230 235240 Phe Leu Thr Phe Leu Asp Arg Met Asp Ser Ser Arg Lys Leu Phe Tyr 245250 255 Val Ile Glu Thr Arg Gln Pro Arg Ala Arg Leu Leu Leu Thr Ala Ala260 265 270 His Leu Leu Phe Val Ala Pro Gln His Asn Gln Ser Glu Ala ThrGly 275 280 285 Ser Thr Ser Gly Gln Ala Leu Phe Ala Ser Asn Val Lys ProGly Gln 290 295 300 Arg Val Tyr Val Leu Gly Glu Gly Gly Gln Gln Leu LeuPro Ala Ser 305 310 315 320 Val His Ser Val Ser Leu Arg Glu Glu Ala SerGly Ala Tyr Ala Pro 325 330 335 Leu Thr Ala Gln Gly Thr Ile Leu Ile AsnArg Val Leu Ala Ser Cys 340 345 350 Tyr Ala Val Ile Glu Glu His Ser TrpAla His Trp Ala Phe Ala Pro 355 360 365 Phe Arg Leu Ala Gln Gly Leu LeuAla Ala Leu Cys Pro Asp Gly Ala 370 375 380 Ile Pro Thr Ala Ala Thr ThrThr Thr Gly Ile His Trp Tyr Ser Arg 385 390 395 400 Leu Leu Tyr Arg IleGly Ser Trp Val Leu Asp Gly Asp Ala Leu His 405 410 415 Pro Leu Gly MetVal Ala Pro Ala Ser 420 425 396 amino acids amino acid linear protein 9Met Ala Leu Pro Ala Ser Leu Leu Pro Leu Cys Cys Leu Ala Leu Leu 1 5 1015 Ala Leu Ser Ala Gln Ser Cys Gly Pro Gly Arg Gly Pro Val Gly Arg 20 2530 Arg Arg Tyr Val Arg Lys Gln Leu Val Pro Leu Leu Tyr Lys Gln Phe 35 4045 Val Pro Ser Met Pro Glu Arg Thr Leu Gly Ala Ser Gly Pro Ala Glu 50 5560 Gly Arg Val Thr Arg Gly Ser Glu Arg Phe Arg Asp Leu Val Pro Asn 65 7075 80 Tyr Asn Pro Asp Ile Ile Phe Lys Asp Glu Glu Asn Ser Gly Ala Asp 8590 95 Arg Leu Met Thr Glu Arg Cys Lys Glu Arg Val Asn Ala Leu Ala Ile100 105 110 Ala Val Met Asn Met Trp Pro Gly Val Arg Leu Arg Val Thr GluGly 115 120 125 Trp Asp Glu Asp Gly His His Ala Gln Asp Ser Leu His TyrGlu Gly 130 135 140 Arg Ala Leu Asp Ile Thr Thr Ser Asp Arg Asp Arg AsnLys Tyr Gly 145 150 155 160 Leu Leu Ala Arg Leu Ala Val Glu Ala Gly PheAsp Trp Val Tyr Tyr 165 170 175 Glu Ser Arg Asn His Ile His Val Ser ValLys Ala Asp Asn Ser Leu 180 185 190 Ala Val Arg Ala Gly Gly Cys Phe ProGly Asn Ala Thr Val Arg Leu 195 200 205 Arg Ser Gly Glu Arg Lys Gly LeuArg Glu Leu His Arg Gly Asp Trp 210 215 220 Val Leu Ala Ala Asp Ala AlaGly Arg Val Val Pro Thr Pro Val Leu 225 230 235 240 Leu Phe Leu Asp ArgAsp Leu Gln Arg Arg Ala Ser Phe Val Ala Val 245 250 255 Glu Thr Glu ArgPro Pro Arg Lys Leu Leu Leu Thr Pro Trp His Leu 260 265 270 Val Phe AlaAla Arg Gly Pro Ala Pro Ala Pro Gly Asp Phe Ala Pro 275 280 285 Val PheAla Arg Arg Leu Arg Ala Gly Asp Ser Val Leu Ala Pro Gly 290 295 300 GlyAsp Ala Leu Gln Pro Ala Arg Val Ala Arg Val Ala Arg Glu Glu 305 310 315320 Ala Val Gly Val Phe Ala Pro Leu Thr Ala His Gly Thr Leu Leu Val 325330 335 Asn Asp Val Leu Ala Ser Cys Tyr Ala Val Leu Glu Ser His Gln Trp340 345 350 Ala His Arg Ala Phe Ala Pro Leu Arg Leu Leu His Ala Leu GlyAla 355 360 365 Leu Leu Pro Gly Gly Ala Val Gln Pro Thr Gly Met His TrpTyr Ser 370 375 380 Arg Leu Leu Tyr Arg Leu Ala Glu Glu Leu Met Gly 385390 395 411 amino acids amino acid linear protein 10 Met Ser Pro Ala TrpLeu Arg Pro Arg Leu Arg Phe Cys Leu Phe Leu 1 5 10 15 Leu Leu Leu LeuLeu Val Pro Ala Ala Arg Gly Cys Gly Pro Gly Arg 20 25 30 Val Val Gly SerArg Arg Arg Pro Pro Arg Lys Leu Val Pro Leu Ala 35 40 45 Tyr Lys Gln PheSer Pro Asn Val Pro Glu Lys Thr Leu Gly Ala Ser 50 55 60 Gly Arg Tyr GluGly Lys Ile Ala Arg Ser Ser Glu Arg Phe Lys Glu 65 70 75 80 Leu Thr ProAsn Tyr Asn Pro Asp Ile Ile Phe Lys Asp Glu Glu Asn 85 90 95 Thr Gly AlaAsp Arg Leu Met Thr Gln Arg Cys Lys Asp Arg Leu Asn 100 105 110 Ser LeuAla Ile Ser Val Met Asn Gln Trp Pro Gly Val Lys Leu Arg 115 120 125 ValThr Glu Gly Arg Asp Glu Asp Gly His His Ser Glu Glu Ser Leu 130 135 140His Tyr Glu Gly Arg Ala Val Asp Ile Thr Thr Ser Asp Arg Asp Arg 145 150155 160 Asn Lys Tyr Gly Leu Leu Ala Arg Leu Ala Val Glu Ala Gly Phe Asp165 170 175 Trp Val Tyr Tyr Glu Ser Lys Ala His Val His Cys Ser Val LysSer 180 185 190 Glu His Ser Ala Ala Ala Lys Thr Gly Gly Cys Phe Pro AlaGly Ala 195 200 205 Gln Val Arg Leu Glu Asn Gly Glu Arg Val Ala Leu SerAla Val Lys 210 215 220 Pro Gly Asp Arg Val Leu Ala Met Gly Glu Asp GlyThr Pro Thr Phe 225 230 235 240 Ser Asp Val Leu Ile Phe Leu Asp Arg GluPro Asn Arg Leu Arg Ala 245 250 255 Phe Gln Val Ile Glu Thr Gln Asp ProPro Arg Arg Leu Ala Leu Thr 260 265 270 Pro Ala His Leu Leu Phe Ile AlaAsp Asn His Thr Glu Pro Ala Ala 275 280 285 His Phe Arg Ala Thr Phe AlaSer His Val Gln Pro Gly Gln Tyr Val 290 295 300 Leu Val Ser Gly Val ProGly Leu Gln Pro Ala Arg Val Ala Ala Val 305 310 315 320 Ser Thr His ValAla Leu Gly Ser Tyr Ala Pro Leu Thr Arg His Gly 325 330 335 Thr Leu ValVal Glu Asp Val Val Ala Ser Cys Phe Ala Ala Val Ala 340 345 350 Asp HisHis Leu Ala Gln Leu Ala Phe Trp Pro Leu Arg Leu Phe Pro 355 360 365 SerLeu Ala Trp Gly Ser Trp Thr Pro Ser Glu Gly Val His Ser Tyr 370 375 380Pro Gln Met Leu Tyr Arg Leu Gly Arg Leu Leu Leu Glu Glu Ser Thr 385 390395 400 Phe His Pro Leu Gly Met Ser Gly Ala Gly Ser 405 410 437 aminoacids amino acid linear protein 11 Met Leu Leu Leu Leu Ala Arg Cys PheLeu Val Ile Leu Ala Ser Ser 1 5 10 15 Leu Leu Val Cys Pro Gly Leu AlaCys Gly Pro Gly Arg Gly Phe Gly 20 25 30 Lys Arg Arg His Pro Lys Lys LeuThr Pro Leu Ala Tyr Lys Gln Phe 35 40 45 Ile Pro Asn Val Ala Glu Lys ThrLeu Gly Ala Ser Gly Arg Tyr Glu 50 55 60 Gly Lys Ile Thr Arg Asn Ser GluArg Phe Lys Glu Leu Thr Pro Asn 65 70 75 80 Tyr Asn Pro Asp Ile Ile PheLys Asp Glu Glu Asn Thr Gly Ala Asp 85 90 95 Arg Leu Met Thr Gln Arg CysLys Asp Lys Leu Asn Ala Leu Ala Ile 100 105 110 Ser Val Met Asn Gln TrpPro Gly Val Arg Leu Arg Val Thr Glu Gly 115 120 125 Trp Asp Glu Asp GlyHis His Ser Glu Glu Ser Leu His Tyr Glu Gly 130 135 140 Arg Ala Val AspIle Thr Thr Ser Asp Arg Asp Arg Ser Lys Tyr Gly 145 150 155 160 Met LeuAla Arg Leu Ala Val Glu Ala Gly Phe Asp Trp Val Tyr Tyr 165 170 175 GluSer Lys Ala His Ile His Cys Ser Val Lys Ala Glu Asn Ser Val 180 185 190Ala Ala Lys Ser Gly Gly Cys Phe Pro Gly Ser Ala Thr Val His Leu 195 200205 Glu Gln Gly Gly Thr Lys Leu Val Lys Asp Leu Arg Pro Gly Asp Arg 210215 220 Val Leu Ala Ala Asp Asp Gln Gly Arg Leu Leu Tyr Ser Asp Phe Leu225 230 235 240 Thr Phe Leu Asp Arg Asp Glu Gly Ala Lys Lys Val Phe TyrVal Ile 245 250 255 Glu Thr Leu Glu Pro Arg Glu Arg Leu Leu Leu Thr AlaAla His Leu 260 265 270 Leu Phe Val Ala Pro His Asn Asp Ser Gly Pro ThrPro Gly Pro Ser 275 280 285 Ala Leu Phe Ala Ser Arg Val Arg Pro Gly GlnArg Val Tyr Val Val 290 295 300 Ala Glu Arg Gly Gly Asp Arg Arg Leu LeuPro Ala Ala Val His Ser 305 310 315 320 Val Thr Leu Arg Glu Glu Glu AlaGly Ala Tyr Ala Pro Leu Thr Ala 325 330 335 His Gly Thr Ile Leu Ile AsnArg Val Leu Ala Ser Cys Tyr Ala Val 340 345 350 Ile Glu Glu His Ser TrpAla His Arg Ala Phe Ala Pro Phe Arg Leu 355 360 365 Ala His Ala Leu LeuAla Ala Leu Ala Pro Ala Arg Thr Asp Gly Gly 370 375 380 Gly Gly Gly SerIle Pro Ala Ala Gln Ser Ala Thr Glu Ala Arg Gly 385 390 395 400 Ala GluPro Thr Ala Gly Ile His Trp Tyr Ser Gln Leu Leu Tyr His 405 410 415 IleGly Thr Trp Leu Leu Asp Ser Glu Thr Met His Pro Leu Gly Met 420 425 430Ala Val Lys Ser Ser 435 418 amino acids amino acid linear protein 12 MetArg Leu Leu Thr Arg Val Leu Leu Val Ser Leu Leu Thr Leu Ser 1 5 10 15Leu Val Val Ser Gly Leu Ala Cys Gly Pro Gly Arg Gly Tyr Gly Arg 20 25 30Arg Arg His Pro Lys Lys Leu Thr Pro Leu Ala Tyr Lys Gln Phe Ile 35 40 45Pro Asn Val Ala Glu Lys Thr Leu Gly Ala Ser Gly Arg Tyr Glu Gly 50 55 60Lys Ile Thr Arg Asn Ser Glu Arg Phe Lys Glu Leu Thr Pro Asn Tyr 65 70 7580 Asn Pro Asp Ile Ile Phe Lys Asp Glu Glu Asn Thr Gly Ala Asp Arg 85 9095 Leu Met Thr Gln Arg Cys Lys Asp Lys Leu Asn Ser Leu Ala Ile Ser 100105 110 Val Met Asn His Trp Pro Gly Val Lys Leu Arg Val Thr Glu Gly Trp115 120 125 Asp Glu Asp Gly His His Phe Glu Glu Ser Leu His Tyr Glu GlyArg 130 135 140 Ala Val Asp Ile Thr Thr Ser Asp Arg Asp Lys Ser Lys TyrGly Thr 145 150 155 160 Leu Ser Arg Leu Ala Val Glu Ala Gly Phe Asp TrpVal Tyr Tyr Glu 165 170 175 Ser Lys Ala His Ile His Cys Ser Val Lys AlaGlu Asn Ser Val Ala 180 185 190 Ala Lys Ser Gly Gly Cys Phe Pro Gly SerAla Leu Val Ser Leu Gln 195 200 205 Asp Gly Gly Gln Lys Ala Val Lys AspLeu Asn Pro Gly Asp Lys Val 210 215 220 Leu Ala Ala Asp Ser Ala Gly AsnLeu Val Phe Ser Asp Phe Ile Met 225 230 235 240 Phe Thr Asp Arg Asp SerThr Thr Arg Arg Val Phe Tyr Val Ile Glu 245 250 255 Thr Gln Glu Pro ValGlu Lys Ile Thr Leu Thr Ala Ala His Leu Leu 260 265 270 Phe Val Leu AspAsn Ser Thr Glu Asp Leu His Thr Met Thr Ala Ala 275 280 285 Tyr Ala SerSer Val Arg Ala Gly Gln Lys Val Met Val Val Asp Asp 290 295 300 Ser GlyGln Leu Lys Ser Val Ile Val Gln Arg Ile Tyr Thr Glu Glu 305 310 315 320Gln Arg Gly Ser Phe Ala Pro Val Thr Ala His Gly Thr Ile Val Val 325 330335 Asp Arg Ile Leu Ala Ser Cys Tyr Ala Val Ile Glu Asp Gln Gly Leu 340345 350 Ala His Leu Ala Phe Ala Pro Ala Arg Leu Tyr Tyr Tyr Val Ser Ser355 360 365 Phe Leu Ser Pro Lys Thr Pro Ala Val Gly Pro Met Arg Leu TyrAsn 370 375 380 Arg Arg Gly Ser Thr Gly Thr Pro Gly Ser Cys His Gln MetGly Thr 385 390 395 400 Trp Leu Leu Asp Ser Asn Met Leu His Pro Leu GlyMet Ser Val Asn 405 410 415 Ser Ser 475 amino acids amino acid linearprotein 13 Met Leu Leu Leu Ala Arg Cys Leu Leu Leu Val Leu Val Ser SerLeu 1 5 10 15 Leu Val Cys Ser Gly Leu Ala Cys Gly Pro Gly Arg Gly PheGly Lys 20 25 30 Arg Arg His Pro Lys Lys Leu Thr Pro Leu Ala Tyr Lys GlnPhe Ile 35 40 45 Pro Asn Val Ala Glu Lys Thr Leu Gly Ala Ser Gly Arg TyrGlu Gly 50 55 60 Lys Ile Ser Arg Asn Ser Glu Arg Phe Lys Glu Leu Thr ProAsn Tyr 65 70 75 80 Asn Pro Asp Ile Ile Phe Lys Asp Glu Glu Asn Thr GlyAla Asp Arg 85 90 95 Leu Met Thr Gln Arg Cys Lys Asp Lys Leu Asn Ala LeuAla Ile Ser 100 105 110 Val Met Asn Gln Trp Pro Gly Val Lys Leu Arg ValThr Glu Gly Trp 115 120 125 Asp Glu Asp Gly His His Ser Glu Glu Ser LeuHis Tyr Glu Gly Arg 130 135 140 Ala Val Asp Ile Thr Thr Ser Asp Arg AspArg Ser Lys Tyr Gly Met 145 150 155 160 Leu Ala Arg Leu Ala Val Glu AlaGly Phe Asp Trp Val Tyr Tyr Glu 165 170 175 Ser Lys Ala His Ile His CysSer Val Lys Ala Glu Asn Ser Val Ala 180 185 190 Ala Lys Ser Gly Gly CysPhe Pro Gly Ser Ala Thr Val His Leu Glu 195 200 205 Gln Gly Gly Thr LysLeu Val Lys Asp Leu Ser Pro Gly Asp Arg Val 210 215 220 Leu Ala Ala AspAsp Gln Gly Arg Leu Leu Tyr Ser Asp Phe Leu Thr 225 230 235 240 Phe LeuAsp Arg Asp Asp Gly Ala Lys Lys Val Phe Tyr Val Ile Glu 245 250 255 ThrArg Glu Pro Arg Glu Arg Leu Leu Leu Thr Ala Ala His Leu Leu 260 265 270Phe Val Ala Pro His Asn Asp Ser Ala Thr Gly Glu Pro Glu Ala Ser 275 280285 Ser Gly Ser Gly Pro Pro Ser Gly Gly Ala Leu Gly Pro Arg Ala Leu 290295 300 Phe Ala Ser Arg Val Arg Pro Gly Gln Arg Val Tyr Val Val Ala Glu305 310 315 320 Arg Asp Gly Asp Arg Arg Leu Leu Pro Ala Ala Val His SerVal Thr 325 330 335 Leu Ser Glu Glu Ala Ala Gly Ala Tyr Ala Pro Leu ThrAla Gln Gly 340 345 350 Thr Ile Leu Ile Asn Arg Val Leu Ala Ser Cys TyrAla Val Ile Glu 355 360 365 Glu His Ser Trp Ala His Arg Ala Phe Ala ProPhe Arg Leu Ala His 370 375 380 Ala Leu Leu Ala Ala Leu Ala Pro Ala ArgThr Asp Arg Gly Gly Asp 385 390 395 400 Ser Gly Gly Gly Asp Arg Gly GlyGly Gly Gly Arg Val Ala Leu Thr 405 410 415 Ala Pro Gly Ala Ala Asp AlaPro Gly Ala Gly Ala Thr Ala Gly Ile 420 425 430 His Trp Tyr Ser Gln LeuLeu Tyr Gln Ile Gly Thr Trp Leu Leu Asp 435 440 445 Ser Glu Ala Leu HisPro Leu Gly Met Ala Val Lys Ser Ser Xaa Ser 450 455 460 Arg Gly Ala GlyGly Gly Ala Arg Glu Gly Ala 465 470 475 313 amino acids amino acidlinear protein 14 Arg Arg Leu Met Thr Gln Arg Cys Lys Asp Arg Leu AsnSer Leu Ala 1 5 10 15 Ile Ser Val Met Asn Gln Trp Pro Gly Val Lys LeuArg Val Thr Glu 20 25 30 Gly Trp Asp Glu Asp Gly His His Ser Glu Glu SerLeu His Tyr Glu 35 40 45 Gly Arg Ala Val Asp Ile Thr Thr Ser Asp Arg AspArg Asn Lys Tyr 50 55 60 Gly Leu Leu Ala Arg Leu Ala Val Glu Ala Gly PheAsp Trp Val Tyr 65 70 75 80 Tyr Glu Ser Lys Ala His Val His Cys Ser ValLys Ser Glu His Ser 85 90 95 Ala Ala Ala Lys Thr Gly Gly Cys Phe Pro AlaGly Ala Gln Val Arg 100 105 110 Leu Glu Ser Gly Ala Arg Val Ala Leu SerAla Val Arg Pro Gly Asp 115 120 125 Arg Val Leu Ala Met Gly Glu Asp GlySer Pro Thr Phe Ser Asp Val 130 135 140 Leu Ile Phe Leu Asp Arg Glu ProHis Arg Leu Arg Ala Phe Gln Val 145 150 155 160 Ile Glu Thr Gln Asp ProPro Arg Arg Leu Ala Leu Thr Pro Ala His 165 170 175 Leu Leu Phe Thr AlaAsp Asn His Thr Glu Pro Ala Ala Arg Phe Arg 180 185 190 Ala Thr Phe AlaSer His Val Gln Pro Gly Gln Tyr Val Leu Val Ala 195 200 205 Gly Val ProGly Leu Gln Pro Ala Arg Val Ala Ala Val Ser Thr His 210 215 220 Val AlaLeu Gly Ala Tyr Ala Pro Leu Thr Lys His Gly Thr Leu Val 225 230 235 240Val Glu Asp Val Val Ala Ser Cys Phe Ala Ala Val Ala Asp His His 245 250255 Leu Ala Gln Leu Ala Phe Trp Pro Leu Arg Leu Phe His Ser Leu Ala 260265 270 Trp Gly Ser Trp Thr Pro Gly Glu Gly Val His Trp Tyr Pro Gln Leu275 280 285 Leu Tyr Arg Leu Gly Arg Leu Leu Leu Glu Glu Gly Ser Phe HisPro 290 295 300 Leu Gly Met Ser Gly Ala Gly Ser Xaa 305 310 64 aminoacids amino acid linear peptide internal 15 Gln Arg Cys Lys Asp Lys LeuAsn Ser Leu Ala Ile Ser Val Met Asn 1 5 10 15 His Trp Pro Gly Val LysLeu Arg Val Thr Glu Gly Trp Asp Glu Asp 20 25 30 Gly His His Phe Glu GluSer Leu His Tyr Glu Gly Arg Ala Val Asp 35 40 45 Ile Thr Thr Ser Asp ArgAsp Lys Ser Lys Tyr Gly Thr Leu Ser Arg 50 55 60 65 amino acids aminoacid linear peptide internal 16 Gln Arg Cys Lys Glu Lys Leu Asn Ser LeuAla Ile Ser Val Met Asn 1 5 10 15 Met Trp Pro Gly Val Lys Leu Arg ValThr Glu Gly Trp Asp Glu Asp 20 25 30 Gly Asn His Phe Glu Asp Ser Leu HisTyr Glu Gly Arg Ala Val Asp 35 40 45 Ile Thr Thr Ser Ser Asp Arg Asp ArgAsn Lys Tyr Gly Met Phe Ala 50 55 60 Arg 65 64 amino acids amino acidlinear peptide internal 17 Gln Arg Cys Lys Asp Lys Leu Asn Ser Leu AlaIle Ser Val Met Asn 1 5 10 15 Leu Trp Pro Gly Val Lys Leu Arg Val ThrGlu Gly Trp Asp Glu Asp 20 25 30 Gly Leu His Ser Glu Glu Ser Leu His TyrGlu Gly Arg Ala Val Asp 35 40 45 Ile Thr Thr Ser Asp Arg Asp Arg Asn LysTyr Arg Met Leu Ala Arg 50 55 60 38 base pairs nucleic acid singlelinear cDNA 18 GGAATTCCCA GCAGNTGCTA AAGGAAGCAA GNGCTNAA 38 33 basepairs nucleic acid single linear cDNA 19 TCATCGATGG ACCCAGATCGAAANCCNGCT CTC 33 29 base pairs nucleic acid single linear cDNA 20GCTCTAGAGC TCNACNGCNA GANCGTNGC 29 50 base pairs nucleic acid singlelinear cDNA 21 AGCTGTCGAC GCGGCCGCTA CGTAGGTTAC CGACGTCAAG CTTAGATCTC 5050 base pairs nucleic acid single linear cDNA 22 AGCTGAGATC TAAGCTTGACGTCGGTAACC TACGTAGCGG CCGCGTCGAC 50 45 base pairs nucleic acid singlelinear cDNA 23 GATCGGCCAG GCAGGCCTCG CGATATCGTC ACCGCGGTAT TCGAA 45 30base pairs nucleic acid single linear cDNA 24 AGTGCCAGTC GGGGCCCCCAGGGCCGCGCC 30 20 base pairs nucleic acid single linear cDNA 25TACCACAGCG GATGGTTCGG 20 20 base pairs nucleic acid single linear cDNA26 GTGGTGGTTA TGCCGATCGC 20 21 base pairs nucleic acid single linearcDNA 27 TAAGAGGCCT ATAAGAGGCG G 21 20 base pairs nucleic acid singlelinear cDNA 28 AAGTCAGCCC AGAGGAGACT 20 6 amino acids amino acid linearpeptide internal 29 Cys Gly Pro Gly Arg Gly 1 5 29 base pairs nucleicacid single linear cDNA 30 AGCAGNTGCT AAAGGAAGCA AGNGCTNAA 29 24 basepairs nucleic acid single linear cDNA 31 CTCNACNGCN AGANCKNGTN GCNA 2432 base pairs nucleic acid single linear cDNA 32 CTGCAGGGAT CCACCATGCGGCTTTTGACG AG 32 31 base pairs nucleic acid single linear cDNA 33CTGCAGGGAT CCTTATTCCA CACGAGGGAT T 31 471 amino acids amino acid linearpeptide internal 34 Met Asp Asn His Ser Ser Val Pro Trp Ala Ser Ala AlaSer Val Thr 1 5 10 15 Cys Leu Ser Leu Asp Ala Lys Cys His Ser Ser SerSer Ser Ser Ser 20 25 30 Ser Lys Ser Ala Ala Ser Ser Ile Ser Ala Ile ProGln Glu Glu Thr 35 40 45 Gln Thr Met Arg His Ile Ala His Thr Gln Arg CysLeu Ser Arg Leu 50 55 60 Thr Ser Leu Val Ala Leu Leu Leu Ile Val Leu ProMet Val Phe Ser 65 70 75 80 Pro Ala His Ser Cys Gly Pro Gly Arg Gly LeuGly Arg His Arg Ala 85 90 95 Arg Asn Leu Tyr Pro Leu Val Leu Lys Gln ThrIle Pro Asn Leu Ser 100 105 110 Glu Tyr Thr Asn Ser Ala Ser Gly Pro LeuGlu Gly Val Ile Arg Arg 115 120 125 Asp Ser Pro Lys Phe Lys Asp Leu ValPro Asn Tyr Asn Arg Asp Ile 130 135 140 Leu Phe Arg Asp Glu Glu Gly ThrGly Ala Asp Arg Leu Met Ser Lys 145 150 155 160 Arg Cys Lys Glu Lys LeuAsn Val Leu Ala Tyr Ser Val Met Asn Glu 165 170 175 Trp Pro Gly Ile ArgLeu Leu Val Thr Glu Ser Trp Asp Glu Asp Tyr 180 185 190 His His Gly GlnGlu Ser Leu His Tyr Glu Gly Arg Ala Val Thr Ile 195 200 205 Ala Thr SerAsp Arg Asp Gln Ser Lys Tyr Gly Met Leu Ala Arg Leu 210 215 220 Ala ValGlu Ala Gly Phe Asp Trp Val Ser Tyr Val Ser Arg Arg His 225 230 235 240Ile Tyr Cys Ser Val Lys Ser Asp Ser Ser Ile Ser Ser His Val His 245 250255 Gly Cys Phe Thr Pro Glu Ser Thr Ala Leu Leu Glu Ser Gly Val Arg 260265 270 Lys Pro Leu Gly Glu Leu Ser Ile Gly Asp Arg Val Leu Ser Met Thr275 280 285 Ala Asn Gly Gln Ala Val Tyr Ser Glu Val Ile Leu Phe Met AspArg 290 295 300 Asn Leu Glu Gln Met Gln Asn Phe Val Gln Leu His Thr AspGly Gly 305 310 315 320 Ala Val Leu Thr Val Thr Pro Ala His Leu Val SerVal Trp Gln Pro 325 330 335 Glu Ser Gln Lys Leu Thr Phe Val Phe Ala AspArg Ile Glu Glu Lys 340 345 350 Asn Gln Val Leu Val Arg Asp Val Glu ThrGly Glu Leu Arg Pro Gln 355 360 365 Arg Val Val Lys Val Gly Ser Val ArgSer Lys Gly Val Val Ala Pro 370 375 380 Leu Thr Arg Glu Gly Thr Ile ValVal Asn Ser Val Ala Ala Ser Cys 385 390 395 400 Tyr Ala Val Ile Asn SerGln Ser Leu Ala His Trp Gly Leu Ala Pro 405 410 415 Met Arg Leu Leu SerThr Leu Glu Ala Trp Leu Pro Ala Lys Glu Gln 420 425 430 Leu His Ser SerPro Lys Val Val Ser Ser Ala Gln Gln Gln Asn Gly 435 440 445 Ile His TrpTyr Ala Asn Ala Leu Tyr Lys Val Lys Asp Tyr Val Leu 450 455 460 Pro GlnSer Trp Arg His Asp 465 470 73 amino acids amino acid linear peptideinternal 35 Arg Cys Lys Glu Arg Val Asn Ser Leu Ala Ile Ala Val Met HisMet 1 5 10 15 Trp Pro Gly Val Arg Leu Arg Val Thr Glu Gly Trp Asp GluAsp Gly 20 25 30 His His Leu Pro Asp Ser Leu His Tyr Glu Gly Arg Ala LeuAsp Ile 35 40 45 Thr Thr Ser Asp Arg Asp Arg His Lys Tyr Gly Met Leu AlaArg Leu 50 55 60 Ala Val Glu Ala Gly Phe Asp Trp Val 65 70 73 aminoacids amino acid linear peptide internal 36 Arg Cys Lys Asp Lys Leu AsnAla Leu Ala Ile Ser Val Met Asn Gln 1 5 10 15 Trp Pro Gly Val Lys LeuArg Val Thr Glu Gly Trp Asp Glu Asp Gly 20 25 30 His His Ser Glu Glu SerLeu His Tyr Glu Gly Arg Ala Val Asp Ile 35 40 45 Thr Thr Ser Asp Arg AspArg Ser Lys Tyr Gly Met Leu Ala Arg Leu 50 55 60 Ala Val Glu Ala Gly PheAsp Trp Val 65 70 64 amino acids amino acid linear peptide internal 37Lys Arg Cys Lys Glu Lys Leu Asn Val Leu Ala Tyr Ser Val Met Asn 1 5 1015 Glu Trp Pro Gly Ile Arg Leu Val Val Thr Glu Ser Trp Asp Glu Asp 20 2530 Tyr His His Gly Gln Glu Ser Leu His Tyr Glu Gly Arg Ala Val Thr 35 4045 Ile Ala Thr Ser Asp Arg Asp Gln Ser Lys Tyr Gly Met Leu Ala Arg 50 5560 28 base pairs nucleic acid single linear cDNA 38 AAAAGCTTTAYTGYTAYGTN GGNATHGG 28 28 base pairs nucleic acid single linear cDNA 39AAGAATTCTA NGCRTTRTAR TTRTTNGG 28 221 amino acids amino acid linearpeptide internal 40 Cys Gly Pro Gly Arg Gly Xaa Gly Xaa Arg Arg His ProLys Lys Leu 1 5 10 15 Thr Pro Leu Ala Tyr Lys Gln Phe Ile Pro Asn ValAla Glu Lys Thr 20 25 30 Leu Gly Ala Ser Gly Arg Tyr Glu Gly Lys Ile XaaArg Asn Ser Glu 35 40 45 Arg Phe Lys Glu Leu Thr Pro Asn Tyr Asn Pro AspIle Ile Phe Lys 50 55 60 Asp Glu Glu Asn Thr Gly Ala Asp Arg Leu Met ThrGln Arg Cys Lys 65 70 75 80 Asp Lys Leu Asn Xaa Leu Ala Ile Ser Val MetAsn Xaa Trp Pro Gly 85 90 95 Val Xaa Leu Arg Val Thr Glu Gly Trp Asp GluAsp Gly His His Xaa 100 105 110 Glu Glu Ser Leu His Tyr Glu Gly Arg AlaVal Asp Ile Thr Thr Ser 115 120 125 Asp Arg Asp Xaa Ser Lys Tyr Gly XaaLeu Xaa Arg Leu Ala Val Glu 130 135 140 Ala Gly Phe Asp Trp Val Tyr TyrGlu Ser Lys Ala His Ile His Cys 145 150 155 160 Ser Val Lys Ala Glu AsnSer Val Ala Ala Lys Ser Gly Gly Cys Phe 165 170 175 Pro Gly Ser Ala XaaVal Xaa Leu Xaa Xaa Gly Gly Xaa Lys Xaa Val 180 185 190 Lys Asp Leu XaaPro Gly Asp Xaa Val Leu Ala Ala Asp Xaa Xaa Gly 195 200 205 Xaa Leu XaaXaa Ser Asp Phe Xaa Xaa Phe Xaa Asp Arg 210 215 220 167 amino acidsamino acid linear peptide internal 41 Cys Gly Pro Gly Arg Gly Xaa XaaXaa Arg Arg Xaa Xaa Xaa Pro Lys 1 5 10 15 Xaa Leu Xaa Pro Leu Xaa TyrLys Gln Phe Xaa Pro Xaa Xaa Xaa Glu 20 25 30 Xaa Thr Leu Gly Ala Ser GlyXaa Xaa Glu Gly Xaa Xaa Xaa Arg Xaa 35 40 45 Ser Glu Arg Phe Xaa Xaa LeuThr Pro Asn Tyr Asn Pro Asp Ile Ile 50 55 60 Phe Lys Asp Glu Glu Asn XaaGly Ala Asp Arg Leu Met Thr Xaa Arg 65 70 75 80 Cys Lys Xaa Xaa Xaa AsnXaa Leu Ala Ile Ser Val Met Asn Xaa Trp 85 90 95 Pro Gly Val Xaa Leu ArgVal Thr Glu Gly Xaa Asp Glu Asp Gly His 100 105 110 His Xaa Xaa Xaa SerLeu His Tyr Glu Gly Arg Ala Xaa Asp Ile Thr 115 120 125 Thr Ser Asp ArgAsp Xaa Xaa Lys Tyr Gly Xaa Leu Xaa Arg Leu Ala 130 135 140 Val Glu AlaGly Phe Asp Trp Val Tyr Tyr Glu Ser Xaa Xaa His Xaa 145 150 155 160 HisXaa Ser Val Lys Xaa Xaa 165 3900 base pairs nucleic acid both linearcDNA CDS 1..3897 42 ATG GAC CGC GAC AGC CTC CCA CGC GTT CCG GAC ACA CACGGC GAT GTG 48 Met Asp Arg Asp Ser Leu Pro Arg Val Pro Asp Thr His GlyAsp Val 1 5 10 15 GTC GAT GAG AAA TTA TTC TCG GAT CTT TAC ATA CGC ACCAGC TGG GTG 96 Val Asp Glu Lys Leu Phe Ser Asp Leu Tyr Ile Arg Thr SerTrp Val 20 25 30 GAC GCC CAA GTG GCG CTC GAT CAG ATA GAT AAG GGC AAA GCGCGT GGC 144 Asp Ala Gln Val Ala Leu Asp Gln Ile Asp Lys Gly Lys Ala ArgGly 35 40 45 AGC CGC ACG GCG ATC TAT CTG CGA TCA GTA TTC CAG TCC CAC CTCGAA 192 Ser Arg Thr Ala Ile Tyr Leu Arg Ser Val Phe Gln Ser His Leu Glu50 55 60 ACC CTC GGC AGC TCC GTG CAA AAG CAC GCG GGC AAG GTG CTA TTC GTG240 Thr Leu Gly Ser Ser Val Gln Lys His Ala Gly Lys Val Leu Phe Val 6570 75 80 GCT ATC CTG GTG CTG AGC ACC TTC TGC GTC GGC CTG AAG AGC GCC CAG288 Ala Ile Leu Val Leu Ser Thr Phe Cys Val Gly Leu Lys Ser Ala Gln 8590 95 ATC CAC TCC AAG GTG CAC CAG CTG TGG ATC CAG GAG GGC GGC GGG CTG336 Ile His Ser Lys Val His Gln Leu Trp Ile Gln Glu Gly Gly Gly Leu 100105 110 GAG GCG GAA CTG GCC TAC ACA CAG AAG ACG ATC GGC GAG GAC GAG TCG384 Glu Ala Glu Leu Ala Tyr Thr Gln Lys Thr Ile Gly Glu Asp Glu Ser 115120 125 GCC ACG CAT CAG CTG CTC ATT CAG ACG ACC CAC GAC CCG AAC GCC TCC432 Ala Thr His Gln Leu Leu Ile Gln Thr Thr His Asp Pro Asn Ala Ser 130135 140 GTC CTG CAT CCG CAG GCG CTG CTT GCC CAC CTG GAG GTC CTG GTC AAG480 Val Leu His Pro Gln Ala Leu Leu Ala His Leu Glu Val Leu Val Lys 145150 155 160 GCC ACC GCC GTC AAG GTG CAC CTC TAC GAC ACC GAA TGG GGG CTGCGC 528 Ala Thr Ala Val Lys Val His Leu Tyr Asp Thr Glu Trp Gly Leu Arg165 170 175 GAC ATG TGC AAC ATG CCG AGC ACG CCC TCC TTC GAG GGC ATC TACTAC 576 Asp Met Cys Asn Met Pro Ser Thr Pro Ser Phe Glu Gly Ile Tyr Tyr180 185 190 ATC GAG CAG ATC CTG CGC CAC CTC ATT CCG TGC TCG ATC ATC ACGCCG 624 Ile Glu Gln Ile Leu Arg His Leu Ile Pro Cys Ser Ile Ile Thr Pro195 200 205 CTG GAC TGT TTC TGG GAG GGA AGC CAG CTG TTG GGT CCG GAA TCAGCG 672 Leu Asp Cys Phe Trp Glu Gly Ser Gln Leu Leu Gly Pro Glu Ser Ala210 215 220 GTC GTT ATA CCA GGC CTC AAC CAA CGA CTC CTG TGG ACC ACA CTGAAT 720 Val Val Ile Pro Gly Leu Asn Gln Arg Leu Leu Trp Thr Thr Leu Asn225 230 235 240 CCC GCC TCT GTG ATG CAG TAT ATG AAG CAG AAG ATG TCC GAGGAA AAG 768 Pro Ala Ser Val Met Gln Tyr Met Lys Gln Lys Met Ser Glu GluLys 245 250 255 ATC AGC TTC GAC TTC GAG ACC GTG GAG CAG TAC ATG AAG CGTGCG GCC 816 Ile Ser Phe Asp Phe Glu Thr Val Glu Gln Tyr Met Lys Arg AlaAla 260 265 270 ATT GCG AGT GGC TAC ATG GAG AAG CCC TGC CTG AAC CCA CTGAAT CCC 864 Ile Ala Ser Gly Tyr Met Glu Lys Pro Cys Leu Asn Pro Leu AsnPro 275 280 285 AAT TGC CCG GAC ACG GCA CCG AAC AAG AAC AGC ACC CAG CCGCCG GAT 912 Asn Cys Pro Asp Thr Ala Pro Asn Lys Asn Ser Thr Gln Pro ProAsp 290 295 300 GTG GGA GCC ATC CTG TCC GGA GGC TGC TAC GGT TAT GCC GCGAAG CAC 960 Val Gly Ala Ile Leu Ser Gly Gly Cys Tyr Gly Tyr Ala Ala LysHis 305 310 315 320 ATG CAC TGG CCG GAG GAG CTG ATT GTG GGC GGA GCG AAGAGG AAC CGC 1008 Met His Trp Pro Glu Glu Leu Ile Val Gly Gly Ala Lys ArgAsn Arg 325 330 335 AGC GGA CAC TTG AGG AAG GCC CAG GCC CTG CAG TCG GTGGTG CAG CTG 1056 Ser Gly His Leu Arg Lys Ala Gln Ala Leu Gln Ser Val ValGln Leu 340 345 350 ATG ACC GAG AAG GAA ATG TAC GAC CAG TGG CAG GAC AACTAC AAG GTG 1104 Met Thr Glu Lys Glu Met Tyr Asp Gln Trp Gln Asp Asn TyrLys Val 355 360 365 CAC CAT CTT GGA TGG ACG CAG GAG AAG GCA GCG GAG GTTTTG AAC GCC 1152 His His Leu Gly Trp Thr Gln Glu Lys Ala Ala Glu Val LeuAsn Ala 370 375 380 TGG CAG CGC AAC TTT TCG CGG GAG GTG GAA CAG CTG CTACGT AAA CAG 1200 Trp Gln Arg Asn Phe Ser Arg Glu Val Glu Gln Leu Leu ArgLys Gln 385 390 395 400 TCG AGA ATT GCC ACC AAC TAC GAT ATC TAC GTG TTCAGC TCG GCT GCA 1248 Ser Arg Ile Ala Thr Asn Tyr Asp Ile Tyr Val Phe SerSer Ala Ala 405 410 415 CTG GAT GAC ATC CTG GCC AAG TTC TCC CAT CCC AGCGCC TTG TCC ATT 1296 Leu Asp Asp Ile Leu Ala Lys Phe Ser His Pro Ser AlaLeu Ser Ile 420 425 430 GTC ATC GGC GTG GCC GTC ACC GTT TTG TAT GCC TTCTGC ACG CTC CTC 1344 Val Ile Gly Val Ala Val Thr Val Leu Tyr Ala Phe CysThr Leu Leu 435 440 445 CGC TGG AGG GAC CCC GTC CGT GGA CAG AGC AGT GTCGGC GTG GCC GGA 1392 Arg Trp Arg Asp Pro Val Arg Gly Gln Ser Ser Val GlyVal Ala Gly 450 455 460 GTT CTG CTC ATG TGC TTT AGT ACC GCC GCC GGA TTGGGA TTG TCA GCC 1440 Val Leu Leu Met Cys Phe Ser Thr Ala Ala Gly Leu GlyLeu Ser Ala 465 470 475 480 CTG CTC GGT ATC GTT TTC AAT GCC GCC AGC ACCCAG GTG GTT CCG TTT 1488 Leu Leu Gly Ile Val Phe Asn Ala Ala Ser Thr GlnVal Val Pro Phe 485 490 495 TTG GCC CTT GGT CTG GGC GTC GAT CAC ATC TTCATG CTG ACC GCT GCC 1536 Leu Ala Leu Gly Leu Gly Val Asp His Ile Phe MetLeu Thr Ala Ala 500 505 510 TAT GCG GAG AGC AAT CGG CGG GAG CAG ACC AAGCTG ATT CTC AAG AAA 1584 Tyr Ala Glu Ser Asn Arg Arg Glu Gln Thr Lys LeuIle Leu Lys Lys 515 520 525 GTG GGA CCG AGC ATC CTG TTC AGT GCC TGC AGCACC GCA GGA TCC TTC 1632 Val Gly Pro Ser Ile Leu Phe Ser Ala Cys Ser ThrAla Gly Ser Phe 530 535 540 TTT GCG GCC GCC TTT ATT CCG GTG CCG GCT TTGAAG GTA TTC TGT CTG 1680 Phe Ala Ala Ala Phe Ile Pro Val Pro Ala Leu LysVal Phe Cys Leu 545 550 555 560 CAG GCT GCC ATC GTA ATG TGC TCC AAT TTGGCA GCG GCT CTA TTG GTT 1728 Gln Ala Ala Ile Val Met Cys Ser Asn Leu AlaAla Ala Leu Leu Val 565 570 575 TTT CCG GCC ATG ATT TCG TTG GAT CTA CGGAGA CGT ACC GCC GGC AGG 1776 Phe Pro Ala Met Ile Ser Leu Asp Leu Arg ArgArg Thr Ala Gly Arg 580 585 590 GCG GAC ATC TTC TGC TGC TGT TTT CCG GTGTGG AAG GAA CAG CCG AAG 1824 Ala Asp Ile Phe Cys Cys Cys Phe Pro Val TrpLys Glu Gln Pro Lys 595 600 605 GTG GCA CCA CCG GTG CTG CCG CTG AAC AACAAC AAC GGG CGC GGG GCC 1872 Val Ala Pro Pro Val Leu Pro Leu Asn Asn AsnAsn Gly Arg Gly Ala 610 615 620 CGG CAT CCG AAG AGC TGC AAC AAC AAC AGGGTG GCG CTG CCC GCC CAG 1920 Arg His Pro Lys Ser Cys Asn Asn Asn Arg ValAla Leu Pro Ala Gln 625 630 635 640 AAT CCT CTG CTG GAA CAG AGG GCA GACATC CCT GGG AGC AGT CAC TCA 1968 Asn Pro Leu Leu Glu Gln Arg Ala Asp IlePro Gly Ser Ser His Ser 645 650 655 CTG GCG TCC TTC TCT CTG GCA ACA TTCGCC TTT CAG CAC TAC ACT CCC 2016 Leu Ala Ser Phe Ser Leu Ala Thr Phe AlaPhe Gln His Tyr Thr Pro 660 665 670 TTC CTC ATG CGC AGC TGG GTG AAG TTCCTG ACC GTT ATG GGT TTC CTG 2064 Phe Leu Met Arg Ser Trp Val Lys Phe LeuThr Val Met Gly Phe Leu 675 680 685 GCG GCC CTC ATA TCC AGC TTG TAT GCCTCC ACG CGC CTT CAG GAT GGC 2112 Ala Ala Leu Ile Ser Ser Leu Tyr Ala SerThr Arg Leu Gln Asp Gly 690 695 700 CTG GAC ATT ATT GAT CTG GTG CCC AAGGAC AGC AAC GAG CAC AAG TTC 2160 Leu Asp Ile Ile Asp Leu Val Pro Lys AspSer Asn Glu His Lys Phe 705 710 715 720 CTG GAT GCT CAA ACT CGG CTC TTTGGC TTC TAC AGC ATG TAT GCG GTT 2208 Leu Asp Ala Gln Thr Arg Leu Phe GlyPhe Tyr Ser Met Tyr Ala Val 725 730 735 ACC CAG GGC AAC TTT GAA TAT CCCACC CAG CAG CAG TTG CTC AGG GAC 2256 Thr Gln Gly Asn Phe Glu Tyr Pro ThrGln Gln Gln Leu Leu Arg Asp 740 745 750 TAC CAT GAT TCC TTT GTG CGG GTGCCA CAT GTG ATC AAG AAT GAT AAT 2304 Tyr His Asp Ser Phe Val Arg Val ProHis Val Ile Lys Asn Asp Asn 755 760 765 GGT GGA CTG CCG GAC TTC TGG CTGCTG CTC TTC AGC GAG TGG CTG GGT 2352 Gly Gly Leu Pro Asp Phe Trp Leu LeuLeu Phe Ser Glu Trp Leu Gly 770 775 780 AAT CTG CAA AAG ATA TTC GAC GAGGAA TAC CGC GAC GGA CGG CTG ACC 2400 Asn Leu Gln Lys Ile Phe Asp Glu GluTyr Arg Asp Gly Arg Leu Thr 785 790 795 800 AAG GAG TGC TGG TTC CCA AACGCC AGC AGC GAT GCC ATC CTG GCC TAC 2448 Lys Glu Cys Trp Phe Pro Asn AlaSer Ser Asp Ala Ile Leu Ala Tyr 805 810 815 AAG CTA ATC GTG CAA ACC GGCCAT GTG GAC AAC CCC GTG GAC AAG GAA 2496 Lys Leu Ile Val Gln Thr Gly HisVal Asp Asn Pro Val Asp Lys Glu 820 825 830 CTG GTG CTC ACC AAT CGC CTGGTC AAC AGC GAT GGC ATC ATC AAC CAA 2544 Leu Val Leu Thr Asn Arg Leu ValAsn Ser Asp Gly Ile Ile Asn Gln 835 840 845 CGC GCC TTC TAC AAC TAT CTGTCG GCA TGG GCC ACC AAC GCG TCT TCG 2592 Arg Ala Phe Tyr Asn Tyr Leu SerAla Trp Ala Thr Asn Ala Ser Ser 850 855 860 CCT ACG GAG CTT CTC AGG GCAAAT TGT ATC CGG AAC CGC GCC AAC GGA 2640 Pro Thr Glu Leu Leu Arg Ala AsnCys Ile Arg Asn Arg Ala Asn Gly 865 870 875 880 GCT TCT CAG GGC AAA TTGTAT CCG GAA CCG CGC CAG TAT TTT CAC CAA 2688 Ala Ser Gln Gly Lys Leu TyrPro Glu Pro Arg Gln Tyr Phe His Gln 885 890 895 CCC AAC GAG TAC GAT CTTAAG ATA CCC AAG AGT CTG CCA TTG GTC TAC 2736 Pro Asn Glu Tyr Asp Leu LysIle Pro Lys Ser Leu Pro Leu Val Tyr 900 905 910 GCT CAG ATG CCC TTT TACCTC CAC GGA CTA ACA GAT ACC TCG CAG ATC 2784 Ala Gln Met Pro Phe Tyr LeuHis Gly Leu Thr Asp Thr Ser Gln Ile 915 920 925 AAG ACC CTG ATA GGT CATATT CGC GAC CTG AGC GTC AAG TAC GAG GGC 2832 Lys Thr Leu Ile Gly His IleArg Asp Leu Ser Val Lys Tyr Glu Gly 930 935 940 TTC GGC CTG CCC AAC TATCCA TCG GGC ATT CCC TTC ATC TTC TGG GAG 2880 Phe Gly Leu Pro Asn Tyr ProSer Gly Ile Pro Phe Ile Phe Trp Glu 945 950 955 960 CAG TAC ATG ACC CTGCGC TCC TCA CTG GCC ATG ATC CTG GCC TGC GTG 2928 Gln Tyr Met Thr Leu ArgSer Ser Leu Ala Met Ile Leu Ala Cys Val 965 970 975 CTA CTC GCC GCC CTGGTG CTG GTC TCC CTG CTC CTG CTC TCC GTT TGG 2976 Leu Leu Ala Ala Leu ValLeu Val Ser Leu Leu Leu Leu Ser Val Trp 980 985 990 GCC GCC GTT CTC GTGATC CTC AGC GTT CTG GCC TCG CTG GCC CAG ATC 3024 Ala Ala Val Leu Val IleLeu Ser Val Leu Ala Ser Leu Ala Gln Ile 995 1000 1005 TTT GGG GCC ATGACT CTG CTG GGC ATC AAA CTC TCG GCC ATT CCG GCA 3072 Phe Gly Ala Met ThrLeu Leu Gly Ile Lys Leu Ser Ala Ile Pro Ala 1010 1015 1020 GTC ATA CTCATC CTC AGC GTG GGC ATG ATG CTG TGC TTC AAT GTG CTG 3120 Val Ile Leu IleLeu Ser Val Gly Met Met Leu Cys Phe Asn Val Leu 1025 1030 1035 1040 ATATCA CTG GGC TTC ATG ACA TCC GTT GGC AAC CGA CAG CGC CGC GTC 3168 Ile SerLeu Gly Phe Met Thr Ser Val Gly Asn Arg Gln Arg Arg Val 1045 1050 1055CAG CTG AGC ATG CAG ATG TCC CTG GGA CCA CTT GTC CAC GGC ATG CTG 3216 GlnLeu Ser Met Gln Met Ser Leu Gly Pro Leu Val His Gly Met Leu 1060 10651070 ACC TCC GGA GTG GCC GTG TTC ATG CTC TCC ACG TCG CCC TTT GAG TTT3264 Thr Ser Gly Val Ala Val Phe Met Leu Ser Thr Ser Pro Phe Glu Phe1075 1080 1085 GTG ATC CGG CAC TTC TGC TGG CTT CTG CTG GTG GTC TTA TGCGTT GGC 3312 Val Ile Arg His Phe Cys Trp Leu Leu Leu Val Val Leu Cys ValGly 1090 1095 1100 GCC TGC AAC AGC CTT TTG GTG TTC CCC ATC CTA CTG AGCATG GTG GGA 3360 Ala Cys Asn Ser Leu Leu Val Phe Pro Ile Leu Leu Ser MetVal Gly 1105 1110 1115 1120 CCG GAG GCG GAG CTG GTG CCG CTG GAG CAT CCAGAC CGC ATA TCC ACG 3408 Pro Glu Ala Glu Leu Val Pro Leu Glu His Pro AspArg Ile Ser Thr 1125 1130 1135 CCC TCT CCG CTG CCC GTG CGC AGC AGC AAGAGA TCG GGC AAA TCC TAT 3456 Pro Ser Pro Leu Pro Val Arg Ser Ser Lys ArgSer Gly Lys Ser Tyr 1140 1145 1150 GTG GTG CAG GGA TCG CGA TCC TCG CGAGGC AGC TGC CAG AAG TCG CAT 3504 Val Val Gln Gly Ser Arg Ser Ser Arg GlySer Cys Gln Lys Ser His 1155 1160 1165 CAC CAC CAC CAC AAA GAC CTT AATGAT CCA TCG CTG ACG ACG ATC ACC 3552 His His His His Lys Asp Leu Asn AspPro Ser Leu Thr Thr Ile Thr 1170 1175 1180 GAG GAG CCG CAG TCG TGG AAGTCC AGC AAC TCG TCC ATC CAG ATG CCC 3600 Glu Glu Pro Gln Ser Trp Lys SerSer Asn Ser Ser Ile Gln Met Pro 1185 1190 1195 1200 AAT GAT TGG ACC TACCAG CCG CGG GAA CAG CGA CCC GCC TCC TAC GCG 3648 Asn Asp Trp Thr Tyr GlnPro Arg Glu Gln Arg Pro Ala Ser Tyr Ala 1205 1210 1215 GCC CCG CCC CCCGCC TAT CAC AAG GCC GCC GCC CAG CAG CAC CAC CAG 3696 Ala Pro Pro Pro AlaTyr His Lys Ala Ala Ala Gln Gln His His Gln 1220 1225 1230 CAT CAG GGCCCG CCC ACA ACG CCC CCG CCG CCC TTC CCG ACG GCC TAT 3744 His Gln Gly ProPro Thr Thr Pro Pro Pro Pro Phe Pro Thr Ala Tyr 1235 1240 1245 CCG CCGGAG CTG CAG AGC ATC GTG GTG CAG CCG GAG GTG ACG GTG GAG 3792 Pro Pro GluLeu Gln Ser Ile Val Val Gln Pro Glu Val Thr Val Glu 1250 1255 1260 ACGACG CAC TCG GAC AGC AAC ACC ACC AAG GTG ACG GCC ACG GCC AAC 3840 Thr ThrHis Ser Asp Ser Asn Thr Thr Lys Val Thr Ala Thr Ala Asn 1265 1270 12751280 ATC AAG GTG GAG CTG GCC ATG CCC GGC AGG GCG GTG CGC AGC TAT AAC3888 Ile Lys Val Glu Leu Ala Met Pro Gly Arg Ala Val Arg Ser Tyr Asn1285 1290 1295 TTT ACG AGT TAG 3900 Phe Thr Ser 24 base pairs nucleicacid single linear cDNA 43 ACCGAGGGCT GGGACGAAGA TGGC 24 25 base pairsnucleic acid single linear cDNA 44 CGCTCGGTCG TACGGCATGA ACGAC 25 27base pairs nucleic acid single linear cDNA 45 ATGGGGATGT GTGTGTGGTCAAGTGTA 27 25 base pairs nucleic acid single linear cDNA 46 TTCACAGACTCTCAAAGTGT ATTTT 25 18 amino acids amino acid linear peptide internal 47Met Gly Ser Ser His His His His His His Leu Val Pro Arg Gly Ser 1 5 1015 His Met

What is claimed is:
 1. A method for modulating growth, differentiation,or survival of a cell comprising contacting said cell with an effectiveamount of a hedgehog polypeptide.
 2. A method for modulating one or moreof growth, differentiation, or survival of a mammalian cell responsiveto hedgehog induction, comprising treating the cell with an effectiveamount of a hedgehog polypeptide thereby altering, relative to the cellin the absence of hedgehog treatment, at least one of (i) rate ofgrowth, (ii) differentiation, or (iii) survival of the cell.
 3. Themethod of claim 2, which polypeptide mimics the effects of anaturally-occurring hedgehog protein on said cell.
 4. The method ofclaim 2, which polypeptide antagonizes the effects of anaturally-occurring hedgehog protein on said cell.
 5. The method ofclaim 2, which polypeptide comprises an amino acid sequence identical orhomologous to an amino acid sequence designated in one of SEQ ID No:8,SEQ ID No:9, SEQ ID No:10, SEQ ID No:11, SEQ ID No:12, SEQ ID No:13 orSEQ ID No:14.
 6. The method of claim 5, which polypeptide is a bioactivefragment of a hedgehog polypeptide.
 7. The method of claim 2, whichpolypeptide comprises an amino acid sequence identical of homologous toan amino acid sequence designated in SEQ ID No:34.
 8. The method ofclaim 2, wherein the cell is a testicular cell, and the polypeptidemodulates spermatogenesis.
 9. The method of claim 2, wherein the cell isan osteogenic cell, and the polypeptide modulates osteogenesis.
 10. Themethod of claim 2, wherein the cell is a chondrogenic cell, and thepolypeptide modulates chondrogenesis.
 11. The method of claim 2, whereinthe polypeptide modulates the differentiation of neuronal cells.
 12. Themethod of claim 11, which neuronal cells are selected from the groupconsisting of motor neurons, cholinergic neurons, dopanergic neurons,serotenergic neurons, and peptidergic neurons.
 13. The method of claim11, wherein the polypeptide promotes survival of the neuronal cells. 14.A method for modulating, in an animal, cell growth, cell differentiationor cell survival, comprising administering a therapeutically effectiveamount of a hedgehog polypeptide to alter, relative the absence ofhedgehog treatment, at least one of (i) rate of growth, (ii)differentiation, or (iii) survival of one or more cell-types in theanimal.
 15. The method of claim 14, which polypeptide mimics the effectsof a naturally-occurring hedgehog protein on cells in the animal. 16.The method of claim 14, which polypeptide antagonizes the effects of anaturally-occurring hedgehog protein on cells in the animal.
 17. Themethod of claim 14, which polypeptide comprises an amino acid sequenceidentical or homologous to amino acid sequence designated in one of SEQID No:8, SEQ ID No:9, SEQ ID No:10, SEQ ID No:11, SEQ ID No:12, SEQ IDNo:13, SEQ ID No:14, SEQ ID No.34, SEQ ID No.40, SEQ ID No.41, orhomologs thereof.
 18. The method of claim 17, which polypeptide is abioactive fragments of a hedgehog polypeptide.
 19. The method of claim14, which method modulates spermatogenesis in the animal.
 20. The methodof claim 14, which method modulates osteogenesis in the animal.
 21. Themethod of claim 14, which method modulates chondrogenesis in the animal.22. The method of claim 14, which method modulates differentiation ofneuronal cells in the animal.
 23. A method for inducing a cell todifferentiate to a neuronal cell phenotype, comprising contacting saidcell with a hedgehog polypeptide.
 24. The method of claim 23, whichpolypeptide comprises an amino acid sequence identical or homologous toamino acid sequence designated in one of SEQ ID No:8, SEQ ID No:9, SEQID No:10, SEQ ID No:11, SEQ ID No:12, SEQ ID No:13, SEQ ID No:14, SEQ IDNo.34, SEQ ID No.40, SEQ ID No.41, or homologs thereof.
 25. The methodof claim 24, which polypeptide is a bioactive fragment of a hedgehogpolypeptide.
 26. The method of claim 23, wherein said neuronal cellphenotype is selected from the group consisting of motor neurons,cholinergic neurons, dopanergic neurons, serotenergic neurons, andpeptidergic neurons.
 27. A method of modulating skeletogenesiscomprising contacting a target tissue with an effective amount of ahedgehog polypeptide so as to cause one or both of chrondrogenesis andoseteogenesis in the target tissue.
 28. The method of claim 27, whereinsaid target tissue is selected from the group consisting of bone,connective tissue and a combination thereof.
 29. A method for treating adegenerative disorder of the nervous system characterized by neuronalcell death, comprising administering to a patient a therapeuticallyeffective amount of a pharmaceutical preparation of a hedgehogpolypeptide thereby causing, relative to the absence of hedgehogtreatment, prolonged survival of neural cells in said patient.
 30. Themethod of calim 29, wherein said hedgehog polypeptide comprises an aminoacid sequence identical or homologous to a polypeptide selected from thegroup consisting of SEQ ID No:8, SEQ ID No:9, SEQ ID No:10, SEQ IDNo:11, SEQ ID No:12, SEQ ID No:13, and SEQ ID No:14, or is a bioactivefragment thereof.
 31. The method of calim 29, wherein said hedgehogpolypeptide comprises an amino acid designated in SEQ ID No.41.
 32. Themethod of calim 29, wherein said hedgehog polypeptide comprises an aminoacid identical or homologous to SEQ ID No. 34, or a bioactive fragmentthereof.
 33. The method of claim 29, wherein said therapeuticallyeffective amount of hedgehog polypeptide inhibits the de-differentiationof neural cells of said patient.
 34. The method of claim 33, whereinsaid neural cell is a glial cell.
 35. The method of claim 33, whereinsaid neural cell is a nerve cell.
 36. The method of claim 29, whereinsaid degenerative disorder is a neuromuscular disorder.
 37. The methodof claim 29, wherein said degenerative disorder is a autonomic disorder.38. The method of claim 29, wherein said degenerative disorder is acentral nervous system disorder.
 39. The method of claim 29, whereinsaid degenerative disorder is selected from a group consisting ofAlzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis,Pick's disease, Huntington's disease, multiple sclerosis, neuronaldamage resulting from anoxia-ischemia, neuronal damage resulting fromtrauma, and neuronal degeneration associated with a natural agingprocess.
 40. The method of claim 29, further comprising administering tosaid patient a therapeutically effective amount of a growth factorhaving neurotrophic activity.
 41. The method of claim 40, wherein saidgrowth factor is selected from a group consisting of a nerve growthfactor, cilliary neurotrophic growth factor, schwanoma-derived growthfactor, glial growth factor, striatal-derived neuronotrophic factor,platelet-derived growth factor.